U.S. patent application number 10/531106 was filed with the patent office on 2006-10-19 for nucleic acid amplification primers for pcr-based clonality studies.
Invention is credited to Christian Bastard, Paul Anthony Stuart Evans, Ramon Garzia Sanz, Michael Hummel, Michael Kneba, Frances Louise Lavender, Elizabeth Anne Macintyre, Gareth John Morgan, Antonio Parreira, Jesus Fernando San Miquel, Eduardus Maria Dominicus Schuuring, John Lewis Smith, Jacobus Johannes Maria Van Dongen.
Application Number | 20060234234 10/531106 |
Document ID | / |
Family ID | 32094087 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234234 |
Kind Code |
A1 |
Van Dongen; Jacobus Johannes Maria
; et al. |
October 19, 2006 |
Nucleic acid amplification primers for pcr-based clonality
studies
Abstract
The invention relates to PCR-based clonality studies for among
others early diagnosis of lymphoproliferative disorders. Provided
is a set of nucleic acid amplification primers comprising a forward
primer, or a variant thereof, and a reverse primer, or a variant
thereof, capable of amplifying a rearrangement selected from the
group consisting of a VH-JH IGH rearrangement, a DH-JH IGH
rearrangement, a VK-JK IGK rearrangement, a VK/intron-Kde IGK
rearrangement, a V.lamda.-J.lamda. IGL rearrangement, a
V.beta.-J.beta. TCRB rearrangement, a D.beta.-J.beta. TCRB
rearrangement, a V.gamma.-J.gamma. TCRG rearrangement, a
V.delta.-J.delta. TCRD rearrangement, a D.delta.-D.delta. TCRD
rearrangement, a D.delta.-J.delta. TCRD rearrangement, a
V.delta.-D.delta. TCRD rearrangement, or a translocation selected
from t(11; 14)(BCL1-IGH) and t(14;18)(BCL2-IGH). The primers can be
used in PCR-based clonality studies for early diagnosis of
lymphoproliferative disorders and detection of minimal residual
disease (MRD). Also provided is a kit comprising at least one set
of primers of the invention.
Inventors: |
Van Dongen; Jacobus Johannes
Maria; (Rotterdam, NL) ; Schuuring; Eduardus Maria
Dominicus; (Groningen, NL) ; San Miquel; Jesus
Fernando; (Salamanca, ES) ; Garzia Sanz; Ramon;
(Salamanca, ES) ; Parreira; Antonio; (Lisboa,
PT) ; Smith; John Lewis; (Wiltshire, AU) ;
Lavender; Frances Louise; (Hants, GB) ; Morgan;
Gareth John; (Suthe, GB) ; Evans; Paul Anthony
Stuart; (Caudle Hill, GB) ; Kneba; Michael;
(Viel, DE) ; Hummel; Michael; (Berlin, DE)
; Macintyre; Elizabeth Anne; (Cedex, FR) ;
Bastard; Christian; (Ardouval, FR) |
Correspondence
Address: |
James C Weseman;The Law Offices of James C Weseman
Suite 1600
401 West A Street
San Diego
CA
92101
US
|
Family ID: |
32094087 |
Appl. No.: |
10/531106 |
Filed: |
October 13, 2003 |
PCT Filed: |
October 13, 2003 |
PCT NO: |
PCT/NL03/00690 |
371 Date: |
March 3, 2006 |
Current U.S.
Class: |
435/6.12 ;
536/24.3 |
Current CPC
Class: |
C12Q 2600/16 20130101;
C12Q 2600/156 20130101; C12Q 1/6886 20130101; C12Q 1/6883 20130101;
C12Q 2600/112 20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 2002 |
US |
60/417779 |
Claims
1. A set of nucleic amplification primers capable of amplifying a
V.sub.H-J.sub.H IGH rearrangement comprising a forward primer and a
reverse primer, wherein said forward primer is selected from the
V.sub.H family primers shown in FIG. 3B, or a variant thereof, and
wherein said reverse primer is the J.sub.H consensus primer shown
in FIG. 3B, or a variant thereof.
2. A set of nucleic amplification primers capable of amplifying a
D.sub.H-J.sub.H IGH rearrangement comprising a forward primer and a
reverse primer, wherein said forward primer is selected from the
D.sub.H family primers shown in FIG. 4A, or a variant thereof, and
wherein said reverse primer is the J.sub.H consensus primer shown
in FIG. 4A, or a variant thereof.
3. A set of nucleic amplification primers capable of amplifying a
V.sub.K-J.sub.K IGK rearrangement comprising a forward primer and a
reverse primer, wherein said forward primer is selected from the
V.sub.K family primers shown in FIG. 5B, or a variant thereof, and
wherein said reverse primer is a J.sub.K primer shown in FIG. 5B,
or a variant thereof.
4. A set of nucleic amplification primers capable of amplifying a
V.sub.K/intron-Kde IGK rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is selected from
the V.kappa. primers or the INTR primer shown in FIG. 5B, or a
variant thereof, and wherein said reverse primer is the Kde primer
shown in FIG. 5B, or a variant thereof.
5. A set of nucleic amplification primers capable of amplifying a
V.lamda.-J.lamda. IGL rearrangement comprising a forward primer and
a reverse primer, wherein said forward primer is selected from the
V.lamda. primers shown in FIG. 6B, or a variant thereof, and
wherein said reverse primer is the J.lamda. primer shown in FIG.
6B, or a variant thereof.
6. A set of nucleic amplification primers capable of amplifying a
V.beta.-J.beta. TCRB rearrangement comprising a forward primer and
a reverse primer, wherein said forward primer is selected from the
V.beta. family primers shown in FIG. 7B, or a variant thereof, and
wherein said reverse primer is selected from the J.beta.A en
J.beta.B primers shown in FIG. 7B, or a variant thereof.
7. A set of nucleic amplification primers capable of amplifying a
D.beta.-J.beta. TCRB rearrangement comprising a forward primer and
a reverse primer, wherein said forward primer is selected from the
DB primers shown in FIG. 7B, or a variant thereof, and wherein said
reverse primer is selected from the J.beta.A en J.beta.B primers
shown in FIG. 7B, or a variant thereof.
8. A set of nucleic amplification primers capable of amplifying a
V.gamma.-J.gamma. TCRG rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is selected from
the V.gamma. family primers shown in FIG. 8B, or a variant thereof,
and wherein said reverse primer is selected from the J.gamma.
primers shown in FIG. 8B, or a variant thereof.
9. A set of nucleic amplification primers capable of amplifying a
V.delta.-J.delta. TCRD rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is selected from
the V.delta. primers shown in FIG. 9B, or a variant thereof, and
wherein said reverse primer is selected from the J.delta. primers
shown in FIG. 9B, or a variant thereof.
10. A set of nucleic amplification primers capable of amplifying a
D.delta.-D.delta. TCRD rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is the D.delta.2
primer shown in FIG. 9B, or a variant thereof, and wherein said
reverse primer is the D.delta.3 primer shown in FIG. 9B, or a
variant thereof.
11. A set of nucleic amplification primers capable of amplifying a
D.delta.-J.delta. TCRD rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is the D.delta.2
primer shown in FIG. 9B, or a variant thereof, and wherein said
reverse primer is selected from the J.delta. primers shown in FIG.
9B, or a variant thereof.
12. A set of nucleic amplification primers capable of amplifying a
V.delta.-D.delta. TCRD rearrangement comprising a forward primer
and a reverse primer, wherein said forward primer is selected from
the V.delta. primers shown in FIG. 9B, or a variant thereof, and
wherein said reverse primer is the D.delta.3 primer shown in FIG.
9B, or a variant thereof.
13. A set of nucleic amplification primers capable of amplifying a
chromosomal translocation (11;14)(BCL1-IGH) comprising a forward
primer and a reverse primer, wherein said forward primer is the
BCL1/MTC primer as shown in FIG. 10A, or a variant thereof, and
wherein said reverse primer is the JH consensus primer shown in
FIG. 10A, or a variant thereof.
14. A set of nucleic amplification primers capable of amplifying a
chromosomal translocation t(14;18)(BCL2-IGH), comprising a forward
primer and a reverse primer, wherein said forward primer is
selected from the MBR primers, the 3'MBR primers and the mcr
primers shown in FIG. 11A, or a variant thereof, and wherein said
reverse primer is the JH consensus primer shown in FIG. 11A, or a
variant thereof.
15. A set of nucleic amplification primers capable of amplifying
the human TBXAS1 gene comprising a forward and a reverse primer,
wherein said forward primer is the TBXAS1/X9U primer of FIG. 12A,
or a variant thereof, and wherein said reverse primer is the
TBXAS1/X9L primer of FIG. 12A, or a variant thereof.
16. A set of nucleic amplification primers capable of amplifying
the human recombination activating protein (RAG1) gene comprising a
forward and a reverse primer, wherein said forward primer is the
RAG1/X2U primer of FIG. 12A, or a variant thereof, and wherein said
reverse primer is the RAG1/X2L primer of FIG. 12A, or a variant
thereof.
17. A set of nucleic amplification primers capable of amplifying
human promyelocytic leukemia zinc finger protein (PLZF) comprising
a forward and a reverse primer, wherein said forward primer is the
PLZF/X1U primer of FIG. 12A, or a variant thereof, and wherein said
reverse primer is the PLZF/X1L primer of FIG. 12A, or a variant
thereof.
18. A set of nucleic amplification primers capable of amplifying
gene the human AF4 gene (Exon 3) comprising a forward and a reverse
primer, wherein said forward primer is the AF4/X3U primer of FIG.
12A, or a variant thereof, and wherein said reverse primer is the
AF4/X3L primer of FIG. 12A, or a variant thereof.
19. A set of nucleic amplification primers capable of amplifying
gene the human AF4 gene (Exon11) comprising a forward and a reverse
primer, wherein said forward primer is the AF4/X11U primer of FIG.
12A, or a variant thereof, and wherein said reverse primer is the
AF4/X11L primer of FIG. 12A, or a variant thereof.
20. A nucleic acid amplification assay, preferably a PCR assay,
more preferably a multiplex PCR assay, using at least one set of
primers according to any one of claims 1 to 19.
21. A method for detecting a V.sub.H-J.sub.H IGH rearrangement,
comprising using one or more sets of primers according to claim 1
in a nucleic acid amplification assay according to claim 20.
22. A method for detecting a D.sub.H-J.sub.H IGH rearrangement,
comprising using one or more sets of primers according to claim 2
in a nucleic acid amplification assay according to claim 20.
23. A method for detecting a V.sub.K-J.sub.K IGK rearrangement,
comprising using one or more sets of primers according to claim 3
in a nucleic acid amplification assay according to claim 20.
24. A method for detecting a V.sub.K/intron-Kde IGK rearrangement,
comprising using one or more sets of primers according to claim 4
in a nucleic acid amplification assay according to claim 20.
25. A method for detecting a V.lamda.-J.lamda. IGL rearrangement,
comprising using one or more sets of primers according to claim 5
in a nucleic acid amplification assay according to claim 20.
26. A method for detecting a V.beta.-J.beta. TCRB rearrangement,
comprising using one or more sets of primers according to claim 6
in a nucleic acid amplification assay according to claim 20.
27. A method for detecting a D.beta.-J.beta. TCRB rearrangement,
comprising using one or more sets of primers according to claim 7
in a nucleic acid amplification assay according to claim 20.
28. A method for detecting a V.gamma.-J.gamma. TCRG rearrangement,
comprising using one or more sets of primers according to claim 8
in a nucleic acid amplification assay according to claim 20.
29. A method for detecting a V.delta.-J.delta. TCRD rearrangement,
comprising using one or more sets of primers according to claim 9
in a nucleic acid amplification assay according to claim 20.
30. A method for detecting a D.delta.-D.delta. TCRD rearrangement,
comprising using one or more sets of primers according to claim 10
in a nucleic acid amplification assay according to claim 20.
31. A method for detecting a D.delta.-J.delta. TCRD rearrangement,
comprising using one or more sets of primers according to claim 11
in a nucleic acid amplification assay according to claim 20.
32. A method for detecting a V.delta.-D.delta. TCRD rearrangement,
comprising using one or more sets of primers according to claim 12
in a nucleic acid amplification assay according to claim 20.
33. A method for detecting a chromosomal translocation
(11;14)(BCL1-IGH), comprising using one or more sets of primers
according to claim 13 in a nucleic acid amplification assay
according to claim 20.
34. A method for detecting a chromosomal translocation
t(14;18)(BCL2-IGH), comprising using one or more sets of primers
according to claim 14 in a nucleic acid amplification assay
according to claim 20.
35. A method for detecting a gene selected from the group
consisting of the human AF4 gene (exon 3), the human AF4 gene (exon
11), the human PLZF1 gene, the human RAG1 gene and the human TBXAS1
gene, comprising using one or more sets of primers according to any
one of claims 15 to 19 in a nucleic acid amplification assay
according to claim 20.
36. Use of a method according to claim 35 to assess the quality of
a DNA sample extracted from a biological sample, preferably a
paraffin-embedded biological sample.
37. A method for detecting two or more rearrangements, two or more
translocations or at least one rearrangement and at least one
translocation selected from the group consisting of a
V.sub.H-J.sub.H IGH rearrangement, a D.sub.H-J.sub.H IGH
rearrangement, a V.sub.K-J.sub.K IGK rearrangement, a
V.sub.K/intron-Kde IGK rearrangement, a V.lamda.-J.lamda. IGL
rearrangement, a V.beta.-J.beta. TCRB rearrangement, a
D.beta.-J.beta. TCRB rearrangement, a V.gamma.-J.gamma. TCRG
rearrangement, a V.delta.-J.delta. TCRD rearrangement, a
D.delta.-D.delta. TCRD rearrangement, a D.delta.-J.delta. TCRD
rearrangement, a V.delta.-D.delta. TCRD rearrangement, a
t(11;14)(BCL1-IGH) translocation and t(14;18)(BCL2-IGH)
translocation, using at least two sets of primers according to any
one of claims 1 or 14.
38. A method for assessing clonal rearrangements and/or chromosome
aberrations using at least one set of primers according to any one
of claims 1 to 14, and optionally at least one set of primers
according to any one of claims 15 to 19.
39. A method according to claim 38 for the detection of minimal
residual disease (MRD) or for identification of PCR targets to be
used for MRD detection via real-time quantitative PCR.
40. A method according to claim 38 or 39, wherein an amplified
nucleic acid is detected using automated high resolution PCR
fragment analysis.
41. A kit for the detection of at least one rearrangement selected
from the group consisting of a V.sub.H-J.sub.H IGH rearrangement, a
D.sub.H-J.sub.H IGH rearrangement, a V.sub.K-J.sub.K IGK
rearrangement, a V.sub.K/intron-Kde IGK rearrangement, a
V.lamda.-J.lamda. IGL rearrangement, a V.beta.-J.beta. TCRB
rearrangement, a D.beta.-J.beta. TCRB rearrangement, a
V.gamma.-J.gamma. TCRG rearrangement, a V.delta.-J.delta. TCRD
rearrangement, a D.delta.-D.delta. TCRD rearrangement, a
D.delta.-J.delta. TCRD rearrangement, a V.delta.-D.delta. TCRD,
and/or at least one translocation selected from t(11;14)(BCL1-IGH)
and t(14;18)(BCL2-IGH), comprising at least one set of primers
according to any one of claims 1 to 14.
42. A kit according to claim 41, further comprising at least one
set of primers according to any one of claims 15 to 19.
Description
[0001] The present invention relates to PCR-based clonality studies
for among others early diagnosis of lymphoproliferative disorders.
In most patients with suspect lymphoproliferative disorders,
histomorphology or cytomorphology supplemented with immunohistology
or flow cytometric immunophenotyping can discriminate between
malignant and reactive lymphoproliferations. However, in 5 to 10%
of cases, making the diagnosis is more complicated. The diagnosis
of lymphoid malignancies can be supported by clonality assessment
based on the fact that in principle all cells of a malignancy have
a common clonal origin.
[0002] The majority of lymphoid malignancies belongs to the B-cell
lineage (90 to 95%) and only a minority belongs to the T-cell
lineage (5-7%) or NK-cell lineage (<2%). Acute lymphoblastic
leukemias (ALL) are of T-cell origin in 15 to 20% of cases, but in
the group of mature lymphoid leukemias and in non-Hodgkin lymphomas
(NHL) T-cell malignancies are relatively rare, except for specific
subgroups such as cutaneous lymphomas (Table 1). Consequently, the
vast majority of lymphoid malignancies (>98%) contains
identically (clonally) rearranged immunoglobulin (Ig) and/or T-cell
receptor (TCR) genes and in 25 to 30% of cases also well-defined
chromosome aberrations are found, all of which can serve as markers
for clonality..sup.1, 2
[0003] The Ig and TCR gene loci contain many different variable
(V), diversity (D), and joining (J) gene segments, which are
subjected to rearrangement processes during early lymphoid
differentiation..sup.3, 4 The V-D-J rearrangements are mediated via
a recombinase enzyme complex in which the RAG1 and RAG2 proteins
play a key role by recognizing and cutting the DNA at the
recombination signal sequences (RSS), which are located downstream
of the V gene segments, at both sides of the D gene segments, and
upstream of the J gene segments (FIG. 1). Inappropriate RSS reduce
or even completely prevent rearrangement.
[0004] The rearrangement process generally starts with a D to J
rearrangement followed by a V to D-J rearrangement in case of Ig
heavy chain (IGH), TCR beta (TCRB), and TCR delta (TCRD) genes
(FIG. 1) or concerns direct V to J rearrangements in case of Ig
kappa (IGK), Ig lambda (IGL), TCR alpha (TCRA), and TCR gamma
(TCRG) genes. The sequences between rearranging gene segments are
generally deleted in the form of a circular excision product, also
called TCR excision circle (TREC) or B cell receptor excision
circle (BREC) FIG. 1).
[0005] The Ig and TCR gene rearrangements during early lymphoid
differentiation generally follow a hierarchical order. During
B-cell differentiation: first the IGH genes rearrange, then IGK,
potentially resulting in IgH/.kappa. expression or followed by IGK
deletion and IGL rearrangement, potentially followed by IgH/.lamda.
expression..sup.5 This implies that virtually all Ig.lamda..sup.+
B-cells have monoallelic or biallelic IGK gene deletions. During
T-cell differentiation: first the TCRD genes rearrange, then TCRG,
potentially resulting in TCR.gamma..delta. expression or followed
by further TCRB rearrangement and TCRD deletion with subsequent
TCRA rearrangement, potentially followed by TCR.alpha..beta.
expression. The Ig and TCR gene rearrangement patterns in lymphoid
malignancies generally fit with the above-described hierarchical
order, although unusual rearrangement patterns are found as well
particularly in ALL..sup.6
[0006] The many different combinations of V, D, and J gene segments
represent the so-called combinatorial repertoire (Table 2), which
is estimated to be .about.2.times.10.sup.6 for Ig molecules,
.about.3.times.10.sup.6 for TCR.alpha..beta. molecules and
.about.5.times.10.sup.3 for TCR.gamma..delta. molecules. At the
junction sites of the V, D, and J gene segments, deletion and
random insertion of nucleotides occurs during the rearrangement
process, resulting in highly diverse junctional regions, which
significantly contribute to the total repertoire of Ig and TCR
molecules, estimated to be >10.sup.12..sup.5
[0007] Mature B-lymphocytes further extend their Ig repertoire upon
antigen recognition in follicle centers via somatic hypermutation,
a process, leading to affinity maturation of the Ig molecules. The
somatic hypermutation process focuses on the V-(D-)J exon of IGH
and Ig light chain genes and concerns single nucleotide mutations
and sometimes also insertions or deletions of nucleotides.
Somatically-mutated Ig genes are also found in mature B-cell
malignancies of follicular or post-foulicular origin..sup.7
[0008] Functionally rearranged Ig and TCR genes result in surface
membrane expression of Ig, TCR.alpha..beta., or TCR.gamma..delta.
molecules. Based on the concept that only a single type of Ig or
TCR molecule is expressed by a lymphocyte or lymphocyte clone, the
clonally rearranged genes of mature lymphoid malignancies might be
detectable at the protein level. Detection of single Ig light chain
expression (Ig.kappa. or Ig.lamda.) has for a long time been used
to discriminate between reactive (polyclonal) B-lymphocytes (normal
Ig.kappa./Ig.lamda. ratio: 0.7-2.8) versus aberrant (clonal)
B-lymphocytes with Ig.kappa./Ig.lamda. ratios of >4.0 or
<0.5..sup.8-10 In the vast majority (>90%) of mature B-cell
malignancies, single Ig light chain expression can support the
clonal origin of the malignancy.
[0009] Also, the development of many different antibodies against
variable domains of the various TCR chains allows detection of
monotypic V.beta., V.gamma. and V.delta. domains, when compared
with appropriate reference values..sup.11-18 In the interpretation
of monotypic V.beta. results using 20 to 25 antibodies against
different V.beta. families (Table 2), one should realize that
clinically-benign clonal TCR.alpha..beta..sup.+ T-cell expansions
(frequently CD8.sup.+) are regularly found in peripheral blood (PB)
of older individuals..sup.13, 17 These clonal T-cell expansions in
PB are however relatively small in size: <40% of PB
T-lymphocytes and <0.5.times.10.sup.6/ml PB..sup.13 It is not
yet clear to what extent such clinically benign T-cell clones can
also be found in lymphoid tissues.
[0010] The results of monotypic V.gamma. and V.delta. domain
expression should be interpreted with caution, because in healthy
individuals a large fraction of normal polyclonal
TCR.gamma..delta..sup.+ T-lymphocytes has been selected for
V.gamma.9-J.gamma.1.2 and V.delta.2-J.delta.1 usage. .sup.18, 19
Consequently, high frequencies of V.gamma.9.sup.+/V.delta.2.sup.+
T-lymphocytes in PB should be regarded as a normal finding, unless
the absolute counts are 1 to 2.times.10.sup.6/ml PB. It should be
noted that most TCR.gamma..delta..sup.+ T-cell malignancies express
V.delta.1 or another non-V.delta.2 gene segment in combination with
a single V.gamma. domain (generally not V.gamma.9).sup.15, 20
[0011] Detection of Ig.kappa. or Ig.lamda. restricted expression or
monotypic V.beta., V.gamma. or V.delta. expression is relatively
easy in flow cytometric studies of PB and bone marrow (BM) samples
of patients with mature B-cell or T-cell leukemias. However, this
appears to be more difficult in tissue samples with suspect
lymphoproliferative disorders that are intermixed with normal
(reactive) lymphocytes.
[0012] In contrast to the antibody-based techniques, molecular
techniques are broadly applicable for detection of clonally
rearranged Ig/TCR genes as well as well-defined chromosome
aberrations. This previously concerned Southern blot analysis, but
nowadays particularly PCR techniques are used. Difficulties in
making a final diagnosis of lymphoid malignancy occur in a
proportion of cases (5 to 10%) despite extensive immunophenotyping.
Therefore, additional (molecular clonality) diagnostics is needed
to generate or to confirm the final diagnosis, such as in case of:
[0013] any suspect B-cell proliferation where morphology and
immunophenotyping are not conclusive; [0014] all suspect T-cell
proliferations (CAUTION: T-cell rich B-NHL); [0015]
lymphoproliferations in immunodeficient patients or transplanted
patients; [0016] evaluation of the clonal relationship between two
lymphoid malignancies in one patient or discrimination between a
relapse and a second malignancy; [0017] further classification of a
malignancy, e.g. via Ig/TCR gene rearrangement patterns or
particular chromosome aberrations; [0018] occasionally: staging of
lymphomas.
[0019] For long time, Southern blot analysis has been the gold
standard technique for molecular clonality studies. Southern
blotting is based on the detection of non-germline ("rearranged")
DNA fragments, obtained after digestion with restriction enzymes.
Well-chosen restriction enzymes (resulting in fragments of 2 to 15
kb) and well-positioned DNA probes (particularly downstream J
segment probes) allow detection of virtually all Ig and TCR gene
rearrangements as well as chromosome aberrations involving J gene
segments..sup.21-28. It should be noted that Southern blot analysis
focuses on the rearrangement diversity of Ig/TCR gene segments and
therefore takes advantage of the combinatorial repertoire.
[0020] Optimal Southern blot results for clonality assessment can
particularly be obtained with the IGH, IGK, and TCRB genes, because
these genes have an extensive combinatorial repertoire as well as a
relatively simple gene structure which can be evaluated with only
one or two DNA probes..sup.22, 24, 28 The IGL and TCRA genes are
more complex and require multiple probe sets..sup.25, 26, 29
Finally, the TCRG and TCRD genes have a limited combinatorial
repertoire, which is less optimal for discrimination between
monoclonality and polyclonality via Southern blot
analysis..sup.20,21
[0021] Despite the high reliability of Southern blot analysis, it
is increasingly replaced by PCR techniques, because of several
inherent disadvantages: Southern blot analysis is time-consuming,
technically demanding, requires 10 to 20 .mu.g of high quality DNA,
and has a limited sensitivity of 5 to 10%..sup.21
[0022] Detection of rearranged Ig/TCR genes and chromosome
aberrations by PCR techniques requires precise knowledge of the
rearranged gene segments in order to design appropriate primers at
opposite sides of the junctional regions and breakpoint fusion
regions, respectively.
[0023] In routine PCR-based clonality studies, the distance between
the primers should be less than 1 kb, preferably less than 500 bp.
This is particularly important for discrimination between PCR
products from monoclonal versus polyclonal Ig/TCR gene
rearrangements, which is based on the diversity of the junctional
regions (diversity in size and composition). So far, mainly IGH and
TCRG gene rearrangements have been used for PCR-based clonality
studies, because of the limited number of primers needed to detect
VH-JH and V.gamma.-J.gamma. rearrangements.
[0024] The main advantages of PCR techniques are their speed, the
low amounts of DNA required, the possibility to use DNA of lower
quality, and the relatively good sensitivity of 1 to 5%, for some
types of rearrangements even <1%. Consequently, PCR techniques
allow the use of small biopsies (e.g. fine needle aspiration
biopsies), or the use of formaldehyde-fixed paraffin-embedded
samples, which generally results in DNA of lower quality. Therefore
also archival material might be used, if needed.
[0025] Molecular clonality studies can be highly informative, but
several limitations and pitfalls might hamper the interpretation of
the results obtained with conventional detection methods:
1. Limited Sensitivity, Related to Normal Polyclonal Background
[0026] The detection limit varies between 1% and 10% (or even 15%),
dependent on the applied technique (Southern blot analysis or PCR
techniques) and dependent on the relative size of the "background"
of normal (polyclonal) B- and T-lymphocytes. A limited sensitivity
might hamper the detection of small clonal cell populations with
less than 5 to 10% clonal lymphoid cells.
2. Clonality is Not Equivalent to Malignancy
[0027] Detection of clonality does not always imply the presence of
a malignancy. Some clinically benign proliferations have a clonal
origin, such as many cases of CD8.sup.+ (or sometimes CD4.sup.+)
T-lymphocytosis, benign monoclonal gammopathies, initial phases of
EBV.sup.+ lymphoproliferations (frequently being oligoclonal) in
immunodeficient patients, and benign cutaneous T-cell
proliferations, such as lymphomatoid papulosis, etc. This implies
that results of molecular clonality studies should always be
interpreted in the context of the clinical, morphological, and
immunophenotypic diagnosis, i.e. in close collaboration with
hematologists, cytomorphologists, pathologists and
immunologists.
3. Ig and TCR Gene Rearrangements are Not Markers for Lineage
[0028] In contrast to the initial assumption, it is now clear for
more than a decade that Ig and TCR gene rearrangements are not
necessarily restricted to B-cell and T-cell lineages, respectively.
Cross-lineage TCR gene rearrangements occur relatively frequently
in immature B-cell malignancies, particularly in precursor-B-ALL
(>90% of cases),.sup.30 but also acute myeloid leukemias (AML)
and mature B-cell malignancies might contain TCR gene
rearrangements..sup.31-33 Albeit at a lower frequency, also
cross-lineage Ig gene rearrangement occur in T-cell malignancies
and AML, mainly involving the Ig heavy chain (IGH) locus..sup.33,
34
[0029] Virtually all (>98%) TCR.alpha..beta..sup.+ T-cell
malignancies have TCRG gene rearrangements (generally biallelic)
and many TCR.gamma..delta..sup.+ T-cell malignancies have TCRB gene
rearrangements, implying that the detection of TCRB or TCRG
rearrangements is not indicative of T-cells of the .alpha..beta. or
.gamma..delta. T-cell lineage, respectively, either. In addition to
these cross-lineage rearrangements, it has been established that
several lymphoid malignancies have unusual Ig/TCR gene
rearrangement patterns. This information is available in detail for
precursor-B-ALL and T-ALL, but not yet for most other lymphoid
malignancies..sup.6
4. Pseudoclonality and Oligoclonality
[0030] The detection of a seemingly clonal or seemingly oligoclonal
lymphoid cell population (pseudoclonality) is rare in Southern blot
analysis, unless genes with a limited combinatorial repertoire are
used, such as TCRG or TCRD. This might result in faint rearranged
bands, e.g. representing V.gamma.9-J.gamma.1.2 or
V.delta.2-J.delta.1 rearrangements derived from antigen-selected
TCR.gamma..delta..sup.+ T-lymphocytes. Yet, this is a well-known
pitfall of Southern blot analysis and will not result in rearranged
bands of high density.
[0031] Pseudoclonality in PCR-based clonality studies is more
difficult to recognize. The high sensitivity of PCR can cause
amplification of the few Ig or TCR gene rearrangements derived from
a limited number of B-cells or T-cells in the studied tissue
sample. Particularly the few reactive (polyclonal) T-cells in a
small needle biopsy or in a B-NHL sample with high tumor load might
result in (oligo)clonal PCR products. Frequently the amount of such
PCR products is limited. This is particularly seen when TCRG genes
are used as PCR target. Duplicate or triplicate PCR analyses
followed by mixing of the obtained PCR products should help to
clarify whether the seemingly clonal PCR products are in fact
derived from different lymphocytes.
[0032] Finally, reactive lymph nodes can show a reduced diversity
of the Ig/TCR repertoire, caused by predominance of several
antigen-selected subclones (oligoclonality). Particularly lymph
nodes or blood samples of patients with an active EBV or CMV
infection can show a restricted TCR repertoire or TCR gene
oligoclonality. Also clinical pictures of immunosuppression are
frequently associated with restricted TCR repertoires, e.g. in
transplant patients or patients with hairy cell leukemia..sup.35
Recovery from transplantation and hematological remission are
followed by restoration of the polyclonal TCR repertoire..sup.36,
37
5. False-Positive Results
[0033] In Southern blot analysis, false-positive results are rare
and can generally be prevented by checking for underdigestion and
by excluding polymorphic restriction sites..sup.21
[0034] False-positive PCR results comprise a serious problem, if no
adequate analysis of the obtained PCR products is performed to
discriminate between monoclonal or polyclonal PCR products. Such
discrimination can be achieved via single-strand conformation
polymorphism (SSCP) analysis,.sup.38 denaturing gradient gel
electrophoresis (DGGE),.sup.39 heteroduplex analysis (HD).sup.40,
41 or GeneScanning (GS)..sup.42, 43 These techniques exploit the
junctional region diversity for discrimination between monoclonal
cells with identical junctional regions and polyclonal cells with
highly diverse junctional regions.
6. False-Negative Results
[0035] False-negative results are rare in Southern blot analysis if
appropriate J gene segment probes are used. Nevertheless, some
uncommon rearrangements (generally non-functional rearrangements)
might be missed, such as V-D rearrangements or deletions of the J
regions. PCR analysis of Ig and TCR genes might be hampered by
false-negative results because of improper annealing of the applied
PCR primers to the rearranged gene segments. This improper primer
annealing can be caused by two different phenomena. Firstly,
precise detection of all different V, D, and J gene segments would
require many different primers (Table 1), which is not feasible in
practice. Consequently, family primers are designed, which
specifically recognize most or all members of a particular V, D, or
J family. Alternatively, consensus primers are used, which are
assumed to recognize virtually all V and J gene segments of the
locus under study. Family primers and particularly consensus
primers are generally optimal for a part of the relevant gene
segments, but show a lower homology (70 to 80%) to other gene
segments. This may eventually lead to false-negative results,
particularly in Ig/TCR genes with many different gene segments. In
TCRG and TCRD genes this problem is minimal, because of their
limited number of different gene segments.
[0036] The second phenomenon is the occurrence of somatic
hypermutations in rearranged Ig genes of follicular and
post-follicular B-cell malignancies, particularly B-cell
malignancies with class-switched IGH genes.
[0037] Sufficient knowledge and experience can prevent the first
four pitfalls, because they mainly concern interpretation problems.
The last two pitfalls concern technical problems, which can be
solved by choosing reliable techniques for PCR product analysis and
by the design of better primer sets.
[0038] Optimization of Southern blot analysis of Ig/TCR genes
during the last ten years has resulted in the selection of reliable
combinations of restriction enzymes (fragments between 2 and 15 kb,
avoiding polymorphic restriction sites) and probes (mainly
downstream of J gene segments). Although Southern blot analysis is
a solid "gold standard" technique, many laboratories have gradually
replaced Southern blot analysis by PCR technology, because PCR is
fast, requires minimal amounts of medium-quality DNA, and has an
overall good sensitivity.
[0039] Despite the obvious advantages, replacement of Southern blot
analysis by PCR techniques for reliable Ig/TCR studies is hampered
by two main technical problems: [0040] false negative results due
to improper primer annealing; [0041] difficulties in discrimination
between monoclonal and polyclonal Ig/TCR gene rearrangements.
[0042] Several individual diagnostic laboratories tried to solve
the problems of the PCR-based clonality studies, but thus far no
reliably standardized PCR protocols were obtained. In contrast,
many different primer sets are being used, which all differ in
their sensitivity and applicability.
[0043] The present invention now provides sets of nucleic acid
amplification primers and standardized PCR protocols for detecting
essentially all relevant Ig and TCR loci and two frequently
occurring chromosome aberrations. The primers sets according to the
invention comprising a forward and a reverse primer are capable of
amplifying clonal rearrangements of the Ig heavy chain genes (IGH),
Ig kappa chain genes (IGK), Ig lamba chain genes (IGL), TCR beta
genes (TCRB), TCR gamma genes (TCRG), and TCR delta genes (TCRD) or
of amplifying chromosomal translocation t(11;14)(BCL1-IGH) and
t(14;18)(BCL2-IGH). The primers of the invention allow that both
complete and incomplete rearrangements are detectable and that gene
segments from different V, (D), and J families can be
recognized.
[0044] Two techniques which can be used in a method of the
invention for discrimination between monoclonal and polyclonal
Ig/TCR gene rearrangements are heteroduplex analysis and
GeneScanning. Heteroduplex analysis uses double-stranded PCR
products and takes advantage of the length and composition of the
junctional regions, whereas in GeneScanning single-stranded PCR
products are separated in a high-resolution gel or polymer
according to their length only (FIG. 2).
[0045] 107 different, specific primers for all the relevant Ig/TCR
loci as well as for t(11;14) (BCL1-IGH) and t(14;18) (BCL2-IGH), or
variants thereof, are provided (see FIGS. 3 to 11). The term
"variant" refers to a primer which differs in 1 to 5 nucleotides,
preferably 1 to 3 nucleotides, from the size and/or position from
the nucleotide of a primer sequence shown, provided that the
nucleotide sequence of said variant primer contains at most 2
mismatches, at most 1 mismatch, most preferably no mismatches with
the target locus. In addition, a variant primer comprises a
(differentially) labeled primer, i.e. a primer having a label that
can be identified or distinguished from other labels by any means,
including the use of an analytical instrument. Examples of
differentially labeled primers are primers provided with a
fluorescent label such as a 6-FAM, HEX, TET or NED dye. Labeled
primers of the invention are particularly advantageous for use in
automated high resolution PCR fragment analysis (Gene Scanning
technology) for detection of PCR products. As is exemplified below,
differentially labeled primers according to the invention allow to
distinguish different PCR amplification products of approximately
the same length (size), preferably using multi-color GeneScanning.
Of course, a variant nucleic acid amplification primer, be it a
forward or a reverse (dye-labeled) primer, should not be capable of
forming dimers with any other (variant) forward and/or reverse
nucleic acid amplification primer that is used in an amplification
reaction, since this can interfere with primer annealing to a
target locus and thus with the amplification of the rearrangement
or translocation of interest.
[0046] In one embodiment, the invention provides a nucleic acid
amplification assay, preferably a PCR assay, using at least one set
of primers according to the invention. Said PCR assay can be a
single (monoplex) or a multiplex PCR. In a preferred embodiment, a
set of primers according to the invention is used in a standardized
multiplex PCR assay, using for example two or more forward primers,
or three or four forward primers, or variants thereof (e.g.
selected from a group of "family primers", for example from the
V.sub.H family primers), together with one or more consensus
reverse primer(s), or variant(s) thereof (e.g. a J.sub.H consensus
primer). The family primers of the invention are designed in such a
way that they recognize most or all gene segments of a particular
family (see Table 2). In a specific embodiment, all 107 primers are
used in only 18 multiplex PCR tubes: 5 for IGH (3x V.sub.H-J.sub.H
and 2x D.sub.H-J.sub.H), 2 for IGK, 1 for IGL, 3 for TCRB (2x
V.beta.-J.beta. and 1x D.beta.-J.beta.), 2 for TCRG, 1 for TCRD, 3
for BCL2-IGH, and 1 for BCL1-IGH (FIGS. 3 to 11). Such an assay
allows assessing clonal rearrangements and/or chromosome
aberrations. Furthermore, it allows detection of a
lymphoproliferative disorder. Multiplex PCR testing of the primers
on about 90 Southern blot defined lymphoproliferations showed that
in more than 95% of the samples the Southern blot and PCR results
were concordant.
[0047] In another embodiment, a method is provided for detecting a
rearrangement, preferably two or more rearrangements, selected from
the group consisting of a V.sub.H-J.sub.H IGH rearrangement, a
D.sub.H-J.sub.H IGH rearrangement, a V.sub.K-J.sub.K IGK
rearrangement, a V.sub.K/intron-Kde IGK rearrangement, a
V.lamda.-J.lamda. IGL rearrangement, a V.beta.-J.beta. TCRB
rearrangement, a D.beta.-J.beta. TCRB rearrangement, a
V.gamma.-J.gamma. TCRG rearrangement, a V.delta.-J.delta. TCRD
rearrangement, a D.delta.-J.delta. TCRD rearrangement, a
D.delta.-D.delta. TCRD rearrangement, and a V.delta.-D.delta. TCRD
rearrangement, using at least one set of primers according to the
invention. Also provided is a method for detecting a
t(11;14)(BCL1-IGH) translocation or a t(14;18)(BCL2-IGH)
translocation, using at least one set of primers according to the
invention. Furthermore, methods are provided for detecting at least
one of the above rearrangements and at least one translocation,
using at least two sets of primers as provided herein.
[0048] In a further aspect, a set of nucleic acid amplification
primers capable of amplifying a human gene selected from the group
consisting of the human AF4 gene (exon 3), the human AF4 gene (exon
11), the human PLZF1 gene, the human RAG1 gene and the human TBXAS1
gene is provided (see FIG. 12). Using one or more of these five
primer sets consisting of a forward primer (or a variant thereof)
and a reverse primer (or a variant thereof in a nucleic acid
amplification assay of the invention, it is possible to detect one
or more "Control Gene(s)" selected from the group consisting of the
human AF4 gene (exon 3), the human AF4 gene (exon 11), the human
PLZF1 gene, the human RAG1 gene and the human TBXAS1 gene. Such a
detection method is advantageously used to assess the quality (e.g.
integrity and amplifiability) of a nucleic acid (DNA) sample
extracted or isolated from a biological sample, for instance DNA
extracted from a paraffin-embedded sample (see Example 10).
[0049] The ability of the different primer sets of the invention to
amplify clonal rearrangements and/or chromosomal aberrations
(translocations) has been tested in many different types of
malignant lymphomas, among which follicular lymphoma, diffuse large
B-cell lymphoma, and multiple myeloma. It was found that a set of
primers is very useful for assessing clonal rearrangements and/or
chromosomal translocations. It appeared that the detection rate of
clonal rearrangements using the multiplex primer tubes according to
the invention is unprecedentedly high, i.e at least 95%.
[0050] Parallel testing of available paraffin-embedded tissues of
the above samples revealed largely identical results, if the DNA
quality of these tissues is sufficiently high, meaning that
fragments of at least 300 bp can be amplified in a
specially-designed control gene PCR.
[0051] The applicability of the developed multiplex PCR assays
according to the invention was evaluated on series of 50 to 100
cases per type of lymphoid malignancy. Following national pathology
panel review, and central pathology panel review in case of
difficulties, all included cases were defined according to the
World Health Organization (WHO) classification. The studied
diagnostic categories included malignancies such as follicular
lymphoma, mantle cell lymphoma, marginal zone lymphoma, diffuse
large B-cell lymphoma, angioimmunoblastic T-cell lymphoma,
peripheral T-cell lymphoma, and anaplastic large cell lymphoma, as
well as reactive lesions. The results show a very high level of
clonality detection, even in entities, which are known to bear
somatic hypermutations such as follicular lymphoma and diffuse
large B-cell lymphoma. Particularly the usage of the three IGH
V.sub.H-J.sub.H tubes, supplemented with the two IGH
D.sub.H-J.sub.H tubes and the two IGK tubes appeared to be highly
efficient in the detection of clonal Ig gene rearrangements. This
high efficiency is obtained by the complementarity of the Ig tubes
as well as by the fact that D.sub.H-J.sub.H and IGK-Kde
rearrangements are not (or rarely) somatically mutated. Such
complementarity was also found for the TCRB and TCRG primers in
case of T-cell malignancies.
[0052] Furthermore, interesting and unexpected rearrangement
patterns, such as unusual cross-lineage rearrangements, were
observed. Remarkably, in about 10% of reactive lesions clonal
rearrangements were detected. These reactive lymphoproliferations
included EBV-related lymphoproliferations and atypical hyperplasias
like Castleman's disease, as well as lesions that were suspicious
for a B- or T-cell clone.
[0053] In a specific embodiment, a method is provided for the
detection of minimal residual disease (MRD). The term minimal
residual disease (MRD) describes the situation in which, after
chemotherapy for acute leukemia (AL), a morphologically normal bone
marrow can still harbor a relevant amount of residual malignant
cells. Detection of minimal residual disease (MRD) is a new
practical tool for a more exact measurement of remission induction
during therapy because leukemic blasts can be detected down to
10.sup.-4-10.sup.-6. Known PCR-based MRD analysis uses clonal
antigen receptor rearrangements detectable in .about.90-95% of the
investigated patient samples. However, amplification of polyclonal
products often leads to false-positive PCR amplicons not suitable
for MRD analysis. The invention now provides a method for the
detection of identically (clonally) rearranged Ig and TCR genes or
detection of well-defined and frequent chromosome aberrations, such
as t(11;14), and t(14;18). Thus, the rearrangements and
translocations detected using a set of primers of the invention not
only serve as markers for clonality at diagnosis, but also as PCR
targets for detection of MRD during follow-up.
[0054] In a further aspect, the invention provides a (diagnostic)
kit for the detection of at least one rearrangement selected from
the group consisting of a V.sub.H-J.sub.H IGH rearrangement, a
D.sub.H-J.sub.H IGH rearrangement, a V.sub.K-J.sub.K IGK
rearrangement, a V.sub.K/intron-Kde IGK rearrangement, a
V.lamda.-J.lamda. IGL rearrangement, a V.beta.-J.beta. TCRB
rearrangement, a D.beta.-J.beta. TCRB rearrangement, a
V.gamma.-J.gamma. TCRG rearrangement, a V.delta.-J.delta. TCRD
rearrangement, a D.delta.-J.delta. TCRD rearrangement, a
D.delta.-D.delta. TCRD rearrangement, a V.delta.-D.delta. TCRD
rearrangement and/or at least one translocation selected from
t(11;14)(BCL1-IGH) and t(14;18)(BCL2-IGH), comprising at least one
set of primers according to the invention. A kit of the invention
is highly suitable for PCR-based clonality diagnostics.
[0055] Optionally, such a kit also comprises at least one set of
primers capable of amplifying a human "control gene" as mentioned
above. Inclusion of one, preferably at least two, more preferably
at least three of these control gene primer sets in a Control Tube
can be helpful in estimating the quality of the DNA sample to be
diagnosed, for instance DNA extracted from-paraffin-embedded
tissue.
[0056] In a further aspect, the invention provides a method for
rapid discrimination of different types of Ig/TCR gene
rearrangements in the same multiplex PCR tube. GeneScanning allows
the application of multiple different fluorochrome-conjugated
primers in a single tube. Such differential labeling of primers can
be used for extra discrimination between different types of Ig or
TCR gene rearrangements.
[0057] Differential labeling of V primers generally has limited
added value, but differential labeling of downstream primers can
support the rapid and easy identification of the type of Ig/TCR
gene rearrangement, which is useful for PCR-based detection of
minimal residual disease..sup.44,45 Labeling of J primers is not
regarded to be informative for IGH (VH-JH or DH-JH), IGK
(V.kappa.-J.kappa.), or IGL (V.lamda.-J.lamda.). For rapid
identification of IGK-Kde rearrangements, it might be interesting
to discriminate between V.kappa.-Kde and intron RSS-Kde
rearrangements by differential labeling of the Kde and intron RSS
primers (see FIG. 5B).
[0058] The most informative multicolor GeneScanning can be designed
for TCR gene rearrangements, facilitating the rapid recognition of
the different types of TCRB, TCRG, and TCRD gene rearrangements.
For example, differential labeling of the J.beta.1 and J.beta.2
primers in TCRB tube A (see FIG. 7B) allows easy identification of
the polyclonal and monoclonal V.beta.-J.beta.1 versus
V.beta.-J.beta.2 rearrangements (FIG. 13A). Differential labeling
of the J.gamma.1.3/2.3 and J.gamma.1.1/2.1 primers (FIG. 8B)
results in easy identification of the different types of TCRG gene
rearrangements (FIG. 13B). Differential labeling of the J.delta.
primers, D.delta.2 primer, and D.delta.3 primer in the TCRD tube
(FIG. 9B) results in easy identification of the most relevant TCRD
gene rearrangements, such as D.delta.2-J.delta., V.delta.-J.delta.,
D.delta.2-D.delta.3, and V.delta.2-D.delta.3 rearrangements (FIG.
13C).
[0059] These multi-color multiplex PCR tubes appear to be easy and
convenient in daily practise of PCR based clonality diagnotics.
LEGENDS TO THE FIGURES
[0060] FIG. 1. Schematic diagram of sequential rearrangement steps,
transcription, and translation of the TCRB gene. In this example
first a D.beta.2 to J.beta.2.3 rearrangement occurs, followed by
V.beta.4 to D.beta.2-J.beta.2.3 rearrangement, resulting in the
formation of a V.beta.4-D.beta.2-J.beta.2.3 coding joint. The
rearranged TCRB gene is transcribed into precursor mRNA, spliced
into mature mRNA, and finally translated into a TCR.beta. protein
chain. The two extrachromosomal TCR excision circles (TRECs) that
are formed during this recombination process are indicated as well;
they contain the D-J signal joint and V-D signal joint,
respectively.
[0061] FIG. 2. Schematic diagram of heteroduplex analysis and
GeneScanning of PCR products, obtained from rearranged Ig and TCR
genes. A. Rearranged Ig and TCR genes (IGH in the example) show
heterogeneous junctional regions with respect to size and
nucleotide composition. Germline nucleotides of V, D, and J gene
segments are given in large capitals and randomly inserted
nucleotides in small capitals. The junctional region heterogeneity
is employed in heteroduplex analysis (size and composition) and
GeneScanning (size only) to discriminate between products derived
from monoclonal and polyclonal lymphoid cell populations. B. In
heteroduplex analysis, PCR products are heat-denatured (5 min,
94.degree. C.) and subsequently rapidly cooled (1 hour, 4.degree.
C.) to induce duplex (homo- or heteroduplex) formation. In cell
samples consisting of clonal lymphoid cells, the PCR products of
rearranged IGH genes give rise to homoduplexes after denaturation
and renaturation, whereas in samples which contain polyclonal
lymphoid cell populations the single-strand PCR fragments will
mainly form heteroduplexes, which result in a background smear of
slowly migrating fragments upon electrophoresis. C. In GeneScanning
fluorochrome-labeled PCR products of rearranged IGH genes are
denatured prior to high-resolution fragment analysis of the
resulting single-stranded fragments. Monoclonal cell samples will
give rise to PCR products of identical size (single peak), whereas
in polyclonal samples many different IGH PCR products will be
formed, which show a characteristic Gaussian size distribution.
[0062] FIG. 3. PCR analysis of IGH (VH-JH) rearrangements. A.
Schematic diagram of IGH gene complex on chromosome band 14q32.3
(adapted from ImMunoGeneTics database)..sup.46, 47 Only
rearrangeable non-polymorphic VH gene segments are included in blue
(functional VH), or in gray (rearrangeable pseudogenes). Recently
discovered (generally truncated) VH pseudogenes are not indicated.
B. Schematic diagram of IGH VH-JH rearrangement with three sets of
VH primers and one JH consensus primer, combined in three multiplex
tubes. The relative position of the VH and JH primers is given
according to their most 5' nucleotide upstream (-) or downstream
(+) of the involved RSS. The VH gene segment used as representative
VH family member for primer design is indicated in parentheses. C,
D, and E. Heteroduplex analysis and GeneScanning of the same
polyclonal and monoclonal cell populations, showing the typical
heteroduplex smears and homoduplex bands (left panels) and the
typical polyclonal Gaussian curves and monoclonal peaks (right
panels). The approximate distribution of the polyclonal Gaussian
curves is indicated in nt.
[0063] FIG. 4. PCR analysis of IGH (DH-JH) rearrangements. A.
Schematic diagram of IGH (DH-JH) rearrangement with seven DH family
primers and one JH consensus primer, divided over two tubes (IGH
tubes D and E). The DH7 (7-27) primer was separated from the other
six DH primers, because the DH7 and JH consensus primer will give a
germline PCR product of 211 nt. The relative position of the DH and
JH primers is given according to their most 5' nucleotide upstream
(-) or downstream (+) of the involved RSS. The DH gene segment used
as representative DH family member for primer design is indicated
in parentheses. B and C. Heteroduplex analysis (left panels) and
GeneScanning (right panels) of the same polyclonal and monoclonal
cell populations. The approximate distribution of the polyclonal
and monoclonal peaks is indicated. The potential background
band/peak in tube D is indicated with an asterisk and is located
outside the expected range of DH-JH rearrangements. The germline
DH7-JH band of tube E is also indicated with an asterisk.
[0064] FIG. 5. PCR analysis of IGK gene rearrangements. A.
Schematic diagram of the IGK gene complex on chromosome band 2p11.2
(adapted from ImMunoGeneTics database)..sup.46,47 Only
rearrangeable non-polymorphic V.kappa. gene segments are indicated
in blue (functional V.kappa.) or in grey (nonfunctional V.kappa.).
The cluster of inverted V.kappa. gene segments (coded with the
letter D) is located .about.800 kb upstream of the non-inverted
V.kappa. gene segments. These upstream V.kappa. gene segments are
presented as a mirrored image to their corresponding non-inverted
counterparts. B. Schematic diagrams of V.kappa.-J.kappa.
rearrangement and the two types of Kde rearrangements (Vk-Kde and
intron RSS-Kde). The relative position of the V.kappa., J.kappa.,
Kde and intron RSS (INTR) primers is given according to their most
5' nucleotide upstream (-) or downstream (+) of the involved RSS.
The V.kappa. gene segment used as representative member of the
V.kappa.1, V.kappa.2, and V.kappa.3 families are indicated in
parentheses. V.kappa.4, V.kappa.5, and V.kappa.7 are single-member
V.kappa. families. The primers are divided over two tubes: tube A
with V.kappa. and J.kappa. primers and tube B with V.kappa., intron
RSS, and Kde primers. C and D. Heteroduplex analysis and
GeneScanning of the same polyclonal and monoclonal cell
populations, showing the typical heteroduplex smears and homoduplex
bands (left panels) and the typical Gaussian curves and monoclonal
peaks (right panels). The approximate distribution of the
polyclonal Gaussian curves is indicated in nt.
[0065] FIG. 6. PCR analysis of IGL gene rearrangements. A.
Schematic diagram of IGL gene complex on chromosome band 22q11.2
(adapted from ImMunoGenetics database)..sup.46, 47 Only
rearrangeable non-polymorphic V.lamda. gene segments are included
in blue (functional V.lamda.) or in grey (nonfunctional V.lamda.)
B. Schematic diagram of V.lamda.-J.lamda. rearrangement with two
V.lamda. family primers and one J.lamda. consensus primer. Only two
V.lamda. primers were designed for V.lamda.1 plus V.lamda.2 and for
V.lamda.3, because these three V.lamda. families cover
approximately 70% of rearrangeable V.lamda. gene segments and
because approximately 90% of all IGL gene rearrangements involve
V.lamda.1, V.lamda.2, or V.lamda.3 gene segments..sup.48 Although
five of the seven J.lamda. gene segments can rearrange, only a
single J.lamda. consensus primer was designed for J.lamda.1,
J.lamda.2, and J.lamda.3, because 98% of all IML gene
rearrangements involve one of these three gene segments..sup.49 The
relative position of the V.lamda. and J.lamda. primers is given
according to their most 5' nucleotide upstream (-) or downstream
(+) of the involved RSS. C. Heteroduplex analysis and GeneScanning
of the same polyclonal and monoclonal cell populations, showing the
typical heteroduplex smears and homoduplex bands (left panel) and
the polyclonal Gaussian curves and monoclonal peaks (right panel).
The approximate position of the polyclonal Gaussian curves is
indicated in nt.
[0066] FIG. 7. PCR analysis of TCRB gene rearrangements. A.
Schematic diagram of the human TCRB locus. The gene segment
designation is according to Arden et al..sup.50 with the
designation according to Rowen et al..sup.51 and Lefranc et
al..sup.48, 47 in parentheses. The figure is adapted from the
international ImMunoGeneTics database..sup.46, 47 Only the
rearrangeable non-polymorphic V.beta. gene segments are depicted in
blue (functional V.beta.), in half blue/half gray (potential
functional, but no protein expression found) and in grey
(non-functional V.beta.). B. Schematic diagram of V.beta.-J.beta.
and D.beta.-J.beta. rearrangements. The 23 V.beta. primers, 13
J.beta. primers and two D.beta. primers are combined in three
tubes: tube A with 23 V.beta. primers and nine J.beta. primers,
tube B with 23 V.beta. primers and four J.beta. primers, and tube C
with two D.beta. primers and 13 J.beta. primers. The 23 V.beta.
primers and the 13 J.beta. primers are aligned in order to obtain
comparably sized PCR products (see panels C and D). The V.beta.
primers cover approximately 90% of all V.beta. gene segments. The
relative position of the V.beta., D.beta., and J.beta. primers is
given according to their most 5' nucleotide upstream (-) or
downstream (+) of the involved RSS. C, D, and E. Heteroduplex
analysis and GeneScanning of the same polyclonal and monoclonal
cell populations, showing the typical heteroduplex smears and
homoduplex bands (left panels) and the typical polyclonal Gaussian
curves and monoclonal peaks (right panels). The approximate
distribution of the polyclonal Gaussian curves is indicated in
nt.
[0067] FIG. 8. PCR analysis of TCRG gene rearrangements. A.
Schematic diagram of the human TCRG locus on chromosome band 7p14.
Only the rearrangeable V.gamma. gene segments are depicted in blue
(functional V.gamma.) or in gray (non-functional V.gamma.). For the
J.gamma. gene segments, both nomenclatures are used..sup.46, 47, 52
B. Schematic diagram of TCRG V.gamma.-J.gamma. rearrangement with
four V.gamma. primers and two J.gamma. primers, which are divided
over two tubes. The relative position of the V.gamma. and J.gamma.
primers is indicated according to their most 5' nucleotide upstream
(-) or downstream (+) of the involved RSS. C and D. Heteroduplex
analysis and GeneScanning of the same polyclonal and monoclonal
cell populations, showing the typical heteroduplex smears and
homoduplex bands (left panels) and the typical polyclonal Gaussian
curves and monoclonal peaks (right panels). The approximate
distribution of the polyclonal Gaussian curves is indicated in
nt.
[0068] FIG. 9. PCR analysis of TCRD gene rearrangements. A.
Schematic diagram of human TCRD locus on chromosome band 14q11.2.
The six "classical" Vd gene segments are indicated in blue,
scattered between the V.alpha. gene segments in black. Since
V.delta.4, V.delta.5, and V.delta.6 are also recognized as V.alpha.
gene segments, their V.alpha. gene code is given in parenthesis. B.
Schematic diagram of V.delta.-J.delta., D.delta.2-J.delta.,
D.delta.2-D.delta.3, and V.delta.-D.delta.3 rearrangements, showing
the positioning of six V.delta., four J.delta., and two D.delta.
primers, all combined in a single tube. The relative position of
the V.delta., D.delta., and J.delta. primers is indicated according
to their most 5' nucleotide upstream (-) or downstream (+) of the
involved RSS. C. Heteroduplex analysis (left panel) and
GeneScanning (right panel) of the same polyclonal and monoclonal
cell populations. The polyclonal cell populations show a vague
smear in heteroduplex analysis and a complex and broad peak pattern
in GeneScanning. The monoclonal bands and peaks are clearly
visible. The approximate position of the PCR products of the
different types of rearrangements in GeneScanning is indicated.
[0069] FIG. 10. Detection of BCL1-IGH rearrangements. A. Schematic
diagram of the CCND1 gene and the BCL1 breakpoint region MTC on
chromosome band 11q13 as well as the JH gene segment on chromosome
band 14q32. For the primer design in the BCL1-MTC region an
artificial BCL1-MTC/JH4 junctional sequence was composed (as
partially reported for JVM2.sup.53): the first 50-nucleotides as
reported by Williams.sup.54 were linked to nucleotide 1-439 from
MTC-sequence present at NCBI (accession-number S77049.sup.55); the
N-region "GCCC" of JVM2.sup.53 was added followed by nucleotide
1921-3182 representing the JH4-JH6 genomic region (accession-number
J00256). B. Agarose gel electrophoresis of a series of BCL1-IGH PCR
products from different MCL patients and the positive control cell
line JVM2. The PCR products differ is size, indicating different
positions of the BCL1-MTC breakpoints. The larger bands of lower
density represent PCR products that extend to the next downstream
germline J.sub.H gene segment.
[0070] FIG. 11. PCR detection of BCL2-IGH rearrangements. A.
Schematic diagram of the BCL2 gene on chromosome band 18q21. The
majority of the BCL2 breakpoints cluster in three regions: MBR, 3'
MBR, and mcr. Consequently, multiplex primers have been designed to
cover the potential breakpoints in these three regions: two MBR
primers, four 3' MBR primers, and three mcr primers. The relative
position of the BCL2 primers is indicated according to their most
5' nucleotide upstream (-) or downstream (+) to the 3' end of BCL2
exon 3 (according to NCBI accession no. AF325194S1), except for two
BCL2-mcr primers; their position is indicated downstream of the
first nucleotide of the AF275873 sequence. B, C, and D. Agarose gel
electrophoresis of PCR products from different FCL patients and
several positive control cell lines (DoHH2, K231, OZ, and SC1).
Panel B and D contain the same samples and show complementarity in
positivity, illustrating that tube C (mcr tube) has added value.
The PCR products differ in size, related to different position of
the BCL2 breakpoints. The larger bands of lower density in the same
lanes represent PCR products that extend to the next downstream
germline JH gene segment or to the next upstream BCL2 primer.
[0071] FIG. 12. Control gene PCR for assessment of amplifiability
and integrity of DNA samples. A. Schematic diagram of five control
genes exons and the five primer sets for obtaining PCR products of
600 bp, 400 bp, 300 bp, 200 bp, and 100 bp. The relative position
of the control gene primers is given according to their most 5'
nucleotide downstream of the 5' splice site of the involved control
gene exon. B. Control gene PCR products of six DNA samples,
separated in a 6% polyacrylamide gel. Two control samples contained
high molecular weight DNA (outer lanes) and four DNA samples were
obtained from paraffin-embedded tissue samples, showing reduced
amplifability (e.g. GBS-4 50 ng versus GBS-4 500 ng) or reduced
integrity of the DNA (PT-4).
[0072] FIG. 13. Multicolor GeneScanning for supporting the rapid
and easy identification of TCR gene rearrangements. A. Two-color
analysis of TCRB tube A with differential labeling of J.beta.1
primers (TET-labeled; green) and J.beta.2 primers (FAM labeled;
blue). The top panel nicely shows the two polyclonal J.beta.1 and
J.beta.2 rearrangement patterns (c.f. FIG. 7C), whereas the other
two panels show clonal J.beta.2 rearrangements. B. Two-color
analysis of TCRG tube A with differential labeling of the
J.gamma.1.3/2.3 primer (FAM-labeled; blue) and the J.gamma.1.1/2.1
(TET-labeled; green). The top panel nicely shows the polyclonal
rearrangement patterns (c.f. FIG. 8C), whereas the other two panels
show clonal J.gamma.1.3/2.3 and clonal J.gamma.1.1/2.1
rearrangements, respectively. C. Three-color analysis of TCRD gene
rearrangements with differential labeling of J.delta. primers
(FAM-labeled; blue), D.delta.2 primer (HEX-labeled; green) and
D.delta.3 primer (NED-labeled; black). Within the complex
rearrangement patterns of the TCRD tube (FIG. 9C), the three-color
analysis allows direct detection of V.delta.-J.delta.
rearrangements (blue peaks), D.delta.2-J.delta. rearrangements
(blue and green peaks, not fully comigrating because of differences
in migration speed of the two fluochromosomes), V.delta.2-D.delta.3
rearrangement (black peaks), and D.delta.2-D.delta.3 rearrangement
(comigrating green and black peaks).
MATERIALS AND METHODS
Selection of PCR Targets: Aiming for Complementarity
[0073] It was decided to aim for the availability of at least one
PCR-detectable clonality target in each lymphoid malignancy. In
mature B-cell malignancies this aim might be hampered by the
occurrence of somatic hypermutations in Ig genes, which are
particularly found in follicular and post-follicular B-cell
malignancies. Therefore it was decided to include PCR targets that
have some degree of complementarity.
[0074] Several rationales were used for target selection: [0075]
IGH genes: not only complete V.sub.H-J.sub.H rearrangements but
also incomplete D.sub.H-J.sub.H rearrangements were included as PCR
targets, because D.sub.H-J.sub.H rearrangements are probably not
affected by somatic hypermutations; [0076] IGK and IGL genes: both
Ig light chain genes were included as PCR targets, because this
increases the chance of finding a PCR-detectable Ig gene
rearrangement in each mature B-cell malignancy; [0077] IGK genes:
not only V.kappa.-J.kappa. rearrangements were included, but also
rearrangements of the kappa deleting element (Kde), because they
occur on one or both alleles in (virtually) all Ig.lamda..sup.+
B-cell malignancies and in one third of Ig.kappa..sup.+ B-cell
malignancies and because Kde rearrangements are probably not
affected by somatic hypermutation; [0078] TCRB genes: both complete
V.beta.-J.beta. and incomplete D.beta.-J.beta. rearrangements,
because complete and incomplete TRCB gene rearrangements occur in
all mature TCR.alpha..beta..sup.+ T-cell malignancies and also in
many TCR.gamma..delta..sup.+ T-cell malignancies; [0079] TCRG
genes: this classical PCR clonality target is useful in all T-cell
malignancies of the TCR.gamma..delta. and the TCR.alpha..beta.
lineage. [0080] TCRD genes: this is a potentially useful target in
immature T-cell malignancies as well as in TCR.gamma..delta..sup.+
T-cell malignancies; [0081] TCRA gene: this gene was not included
as PCR target, because of its high degree of complexity with
.about.50 V and 61 J gene segments. Furthermore, all T-cell
malignancies with TCRA gene rearrangements contain TCRB gene
rearrangements and generally also have TCRG gene rearrangements;
[0082] functional gene segments: most suspect lymphoproliferations
concern mature lymphocytes, which consequently have functional Ig
or TCR gene rearrangements. Therefore PCR primer design aimed at
inclusion of (virtually) all functional Ig/TCR gene segments.
[0083] well-defined chromosome aberrations: t(11;14) with BCL1-IGH
and t(14;18) with BCL2-IGH were included as additional targets,
because these two aberrations are PCR-detectable at relatively high
frequencies in lymphomas i.e. in 30% of mantle cell lymphoma (MCL)
and in 60 to 70% of follicular cell lymphomas (FCL), respectively.
Primer Design for Multiplex PCR
[0084] Precise detection of all V, D, and J gene segments in
rearranged Ig and TCR genes would require many different primers
(Table 2). For some gene complexes this might be possible (e.g.
TCRG and TCRD), but for other loci in practice this is impossible
because of the high number of different gene segments. To solve
this problem, family primers can be designed, which recognize most
or all gene segments of a particular family (Table 2).
Alternatively, consensus primers can be made, which recognize
conserved sequences that occur in many or all involved gene
segments.
[0085] The design of family primers and consensus primers balances
between a limited number of primers and maximal homology with all
relevant gene segments. In this study, we aimed at maximal homology
with all relevant gene segments (particularly functional gene
segments) in order to prevent suboptimal primer annealing, which
might cause false-negative results. Furthermore, we aimed at the
design of specific family primers without cross-annealing to other
families
[0086] In order to limit the number of PCR tubes per locus,
multiplexing of PCR primers became important for practical reasons.
Consequently, special guidelines were developed to ensure maximal
possibilities for designing primers useful in multiplex PCR tubes.
For this purpose dr. W. Rychlick (Molecular Biology Insights,
Cascade, Colo., USA) provided his specially-adapted OLIGO 6.2
software program and supported the development of the guidelines
for optimal primer design.
[0087] The general guidelines for primer design were as follows:
[0088] the position of the primers should be chosen in such a way
that the size of the PCR products would preferably be <300 bp
(preferably 100 to 300 bp) in order to be able to use
paraffin-embedded material; [0089] a minimal distance to the
junctional region of preferaby >10-15 bp should be taken into
acount (in order to avoid false-negativity due to impossibility of
the 3' end of the primer to anneal to the rearranged target because
of nucleotide deletion from the germline sequence); [0090] primers
preferably should not be too long (e.g. <25 nucleotides).
[0091] The following parameters were used for primer design with
the OLIGO 6.2 program: [0092] search for primers should be
performed with moderate stringency; [0093] primer efficiency (PE)
value should preferably be .about.400 (and >630, if the primer
is to be used as consensus primer for other gene segments as well);
[0094] the most stable 3' dimer of upper/upper, lower/lower, or
upper/lower primers should not exceed -4 Kcal (moderate search
strategy); the most stable dimer overall being less important;
[0095] in view of multiplex PCR, the following guidelines were
taken into account: a common primer would have to be designed in
the most consensus region (i.e. high PE in consensus search),
whereas individual primers (family or member) have to be designed
in the least consensus region (i.e. low PE value of that primer for
gene segments that should not be covered) to avoid cross-annealing
to other gene segments and thereby multiple (unwanted) PCR
products. PCR Protocol
[0096] A standardised PCR protocol was developed based on
pre-existing experience from earlier European collaborative
studies. After initial testing and approval, the protocol was
accepted as summarized in Table 3.
Techniques for Analysis of PCR Products Obtained from Ig/TCR Gene
Rearrangements
[0097] The PCR products obtained from Ig and TCR gene
rearrangements have to be analysed for discrimination between
monoclonal lymphoid cells with identical junctional regions and
polyclonal lymphoid cells with highly diverse junctional
regions.
[0098] Based on the combined experience of the participating
laboratories, two techniques were selected: heteroduplex (HD)
analysis and Gene Scanning (GS) analysis. HD analysis uses
double-stranded PCR products and takes advantage of the length and
composition of the junctional regions, whereas in GS
single-stranded PCR products are separated in a high resolution gel
or polymer according to their length only (FIG. 2).
Heteroduplex Analysis of PCR Products
[0099] PCR products obtained with unlabeled primers are denatured
at high temperature (.about.95.degree. C. for 5 min), followed by
rapid random renaturation at low temperature (preferably at
4.degree. C. for 1 hour). This enforced duplex formation results in
many different heteroduplexes with different migration speed in
case of polyclonal lymphoproliferations, but resulting in
homoduplexes with identical rapid migration in case of monoclonal
lymphoproliferations. Electrophoresis of the homoduplexes in a 6%
polyacrylamide gel results in a single band of predictable size,
whereas the heteroduplexes form a smear at a higher position (FIG.
2). The heteroduplex technique is rapid, simple and cheap (see
Table 4 for technical details) and has a detection limit of
.about.5%..sup.40,41 The detection limit is influenced by the
frequency of polyclonal lymphocytes, because the formation of many
heteroduplexes will also consume a part of the monoclonal PCR
products..sup.41
Genescanning Analysis of PCR Products
[0100] The PCR primers for GeneScanning analysis need to be labeled
with a fluorochrome to allow detection of the PCR products with
automated sequencing equipment (FIG. 2).
[0101] The fluorochrome labeled single-strand (denatured) PCR
products are size-separated in a denaturing polyacrylamide
sequencing gel or capillary sequencing polymer and detected via
automated scanning with a laser (see Table 5 for technical
details). This results in a Gausian distribution of multiple peaks,
representing many different PCR products in case of polyclonal
lymphoproliferations, but gives a single peak consisting of one
type of PCR product in case of a fully monoclonal
lymphoproliferation (FIG. 2).
[0102] GeneScanning is rapid and relatively simple, but needs
expensive equipment. GeneScanning is generally more sensitive than
heteroduplex analysis and can reach sensitivities of 0.5 to 1% of
clonal lymphoid cells.
Control Genes and Paraffin-Embedded Tissues
[0103] In several European countries, fresh tissue material is not
easily available for molecular diagnostics such as PCR-based
clonality studies. Therefore one of the aims of the present study
was to develop a strategy for PCR-based clonality studies in
paraffin-embedded tissues.
[0104] To control for the quality and amplifiability of DNA from
paraffin-embedded material, a special multiplex control gene PCR
was developed, resulting in a ladder of five fragments (100 bp, 200
bp, 300 bp, 400 bp, and 600 bp). From 45 of the above described 90
cases sufficient paraffin-embedded tissue was available for DNA
extraction. These DNA samples were tested in parallel to the
freshly-obtained DNA samples, using the Control Gene multiplex tube
as well as the Ig/TCR/BCL1/BCL2 multiplex tubes for clonality
diagnostics (see Example 10).
EXAMPLE 1
Complete IGH Gene Rearrangements: VH-JH
Background
[0105] The functional rearrangement of the IGH gene, first D.sub.H
to J.sub.H and subsequently V to D.sub.H-J.sub.H, is followed by
antibody expression, the hallmark of mature B-cells. The IGH gene
is located on chromosome 14q32.3 in an area covering approximately
1250 kilobases. 46 to 52 functional VH segments (depending on the
individual haplotype) have been identified, which can be grouped
according to their homology in six or seven VH subgroups. In
addition approximately 30 non-functional VH segments have been
described. Furthermore, 27 DH segments and functional six JH
segments have been consistently found (Table 2 and FIG.
3A)..sup.56
[0106] The VH segments contain three framework (FR) and two
complementarity determining regions (CDR) (FIG. 3B). The FRs are
characterized by their similarity among the various VH segments
whereas the CDRs are highly different even within the same VH
family. Furthermore, the CDRs represent the preferred target
sequences for somatic hypermutations in the course of the germinal
center reaction, which increase the variability within those
regions. Although the FRs are usually less affected by somatic
mutations, nucleotide substitutions may also occur within these
regions, especially in B-cells under a heavy mutational
process.
[0107] The highly variable V-D-JH regions can-be amplified by PCR
to detect clonal B-cell populations indicative of the presence of a
malignant B-cell disorder. Clonal B-cells can be discriminated from
polyclonal B-cells (i.e. normal or reactive lymphoid tissues) based
on the identical size and composition of the clonal PCR products as
compared to the many different polyclonal PCR products with a size
range of approximately 60 bp, arranged in a Gaussian distribution.
PCR-based strategies for detection of clonal B-cell populations in
histological sections and cell suspensions have already been
established in the early nineties. However, the initial PCR
protocols used single VH consensus primers which were able to bind
to one of the three framework regions, mainly FR3. Such consensus
primers were not suitable to amplify all VH segments with the same
efficiency leading to non-detectability of a significant number of
clonal rearrangements. In addition, somatic mutations introduced in
the course of the germinal center reaction are not restricted to
the CDRs, but can also occur in FRs, thereby preventing primer
annealing and consequently leading to absence of clonal PCR
products despite the presence of a neoplastic B-cell population.
This is especially true for follicular lymphomas, diffuse large
B-cell lymphomas, and multiple myelomas which usually contain high
numbers of somatic mutations.
[0108] To further increase the detection rate of the IGH PCR,
several attempts have been made to design family-specific primers
to overcome the limitations of consensus primers. However, these
family-specific primers are largely based on the sequences of the
previous consensus primers. Although these PCR strategies have
helped to improve the detection rate, there is still a need of
primer systems which are less sensitive to somatic hypermutations,
thus allowing amplification of (virtually) all possible V-D-JH
rearrangements.
Primer Design
[0109] According to the guidelines of the invention, three sets of
VH primers were designed with the help of the OLIGO-6.2 program
corresponding to the three VH frame work regions (FR1, FR2 and FR3)
(FIG. 3B). Each set of primers consisted of six or seven
oligonucleotides capable to anneal to their corresponding VH
segments (VH.sub.1 to VH.sub.7) with no mismatches for most VH
segments and one or at most two mismatches for some rare VH
segments. The design was such that mismatches would be located at
the very 5'-end of the primer. These VH primer sets were used in
conjunction with a single JH consensus primer, designed to anneal
to the most homologous 3'-end of the six JH segments, approximately
35 bp downstream of the JH RSS. This ensures that all JH segments
are detectable with the same binding efficiency and that the primer
binding will not easily be affected by extensive nucleotide
deletion in the course of the rearrangement process. In addition,
there was no cross-annealing between the VH primers and the JH
primer as evaluated by the OLIGO-6.2 program.
[0110] The JH primer was also designed to be used for amplification
of other PCR targets, such as incomplete DH-JH rearrangements as
well as t(11;14) (BCL1-IGH) and t(14;18) (BCL2-IGH). This allows
the detection of different PCR products by GS analysis employing
the same labeled JH primer.
Results of Initial Testing Phase
[0111] The initial testing of the newly designed VH-JH PCR was done
by separate application of each VH primer together with the JH
primer in an individual PCR. For this purpose, DNA extracted from
B-cell lines as well as well-defined clonal patient samples was
used. Furthermore, clonal rearrangements were tested for
sensitivity by serial dilution in DNA extracted from reactive
tonsils. Clonal control samples were not available for each
possible IGH rearrangement, but all major VH segments and several
rarely rearranged VH segments have been included in the initial
testing phase.
[0112] All primer pairs worked with high efficiency and
sensitivity. The expected clonal VH rearrangements were detectable
and the sensitivity was at least 1% (10.sup.-2). There was no
background within the expected size range and the amplification of
tonsillar DNA gave the expected Gaussian distribution curve. (FIGS.
3C, D, and E)
[0113] Based on these results we started the next phase of the
initial primer testing by combining the VH primers into three sets,
each specific for one of the three framework regions, which were
used together with the common JH primer (FIG. 3B). The results were
the same as those obtained with single primer pairs, but with a
slightly lower sensitivity. In addition, no nonspecific products
were amplified within the expected size range, with the exception
of a 340 bp PCR product which appeared in the FR1 multiplex PCR.
This PCR product was generated irrespective of the source of the
DNA (lymphoid and non-lymphoid) used for PCR, whereas no PCR
product was obtained when no DNA template was applied. Furthermore,
this amplicon was only detectable in heteroduplex analyis, not in
GeneScanning. This indicates that the fluorescent labeled JH primer
was not involved in the generation of this PCR product. Sequence
analysis of this PCR product disclosed a VH4 fragment amplified by
the FR1 VH4 primer in conjunction with the FR1 VH2 primer which
apparently acted as a downstream primer by binding to the intronic
VH4 sequence. This problem could be solved by designing a new FR1
VH2 primer which was located 25 bp upstream to the previous primer
binding site.
Results of General Testing Phase
[0114] The approved IGH PCR was applied to the 90 Southern blot
defined DNA samples, which were derived from well-characterized
cases. Six of the 11 laboratories involved in the general testing
phase performed GS analysis of the PCR products and five performed
HD analysis. In addition several polyclonal as well as monoclonal
samples (cell line DNA) were included as controls. 45 of these
cases displayed dominant PCR products after GS analysis and 40
cases after HD detection, indicating the presence of a monoclonal
B-cell population. The clonal rearrangements were detectable with
all three FR primer sets in 33 of the 45 clonal cases (GS) and in
the remaining 12 with one or two of the three FR primer sets. It
was concluded that most negative results were caused by somatic
hypermutations in the primer binding site, preventing primer
annealing and thus amplification.
[0115] The comparison of the VH-JH PCR results with the Southern
blot results revealed a high degree of concordance. 85% (46 out of
55) and 76% (42 out of 55) of the samples with rearranged VH genes
by Southern blot analysis showed a dominant amplification product
by GS analysis and HD analysis, respectively. Vice versa, all but
two samples harboring germline VH genes by Southern blot displayed
a polyclonal pattern by GS and HD analysis.
Conclusion
[0116] In conclusion, the three multiplex PCRs for detection of
clonal VH-JH rearrangements provide a new and reliable assay to
identify clonal B-cell proliferations. The combined use of
standardized primers in the three different FRs helps to decrease
the rate of false-negative results due to somatic hypermutation in
primer binding sites of the involved VH gene segments.
EXAMPLE 2
Incomplete IGH Gene Rearrangements: D.sub.H-J.sub.H
Background
[0117] The formation of complete V-D-J rearrangements in the IGH
locus on chromosome 14q32.3 is a sequential process that occurs in
two steps: VH coupling is generally preceded by an initial
rearrangement between DH and JH gene segments in early precursor-B
cells (reviewed by.sup.57). In addition to the many distinct VH
gene segments and the six functional JH gene segments (see Example
1), the human IGH locus also contains 27 DH gene segments..sup.58
Based on sequence homology, the 27 DH segments can be grouped into
seven families: DH1 (formerly known as DM), DH2 (DLR), DH3 (DXP),
DH4 (DA), DH5 ([K), DH6 (DN), and DH7 (DQ52); all families comprise
at least four members, except for the seventh which consists of the
single DH7-27 segment just upstream of the JH region (FIG.
3A)..sup.58,59
[0118] Recombination between any of the DH and JH segments will
result in the formation of incomplete DH-JH joints, which can
easily be detected in bone marrow-derived CD10.sup.+/CD19.sup.-
precursor B-cells.sup.60, 61 and hence also in a subset (20-25%) of
precursor B-cell acute lymphoblastic leukemias, which show an
immature genotypes..sup.62 Sequencing revealed a predominance of
DH2 (DH2-2), DH3 (DH3-9), and DH7-27 gene segments in precursor
B-ALL, comprising 36%, 33%, and 19% of all identified segments,
respectively..sup.62
[0119] However, also in mature B-cell malignancies incomplete DH-JH
rearrangements have been reported..sup.61,63 Moreover, even in a
subset of IgH-negative multiple myelomas, which can be considered
as the most mature type of B-lineage malignancy, DH-JH joints were
observed..sup.64 These DH-JH rearrangements were derived from the
non-coding second allele and involved segments from DH1 to DH4
families..sup.64 Based on the description of DH-JH joints in
precursor-B-ALL and multiple myelomas, it is assumed that
incomplete DH-JH rearrangements are also present in other types of
B-cell leukemias and lymphomas. In immature T-cell malignancies
DH-JH couplings have been identified as cross-lineage
rearrangements;.sup.54 interestingly, these almost exclusively
occurred in the more immature non-TCR.alpha..beta..sup.+ T-ALL
subset and mainly involved the more downstream DH6-19 and DH7-27
segments. The latter segment is frequently (up to 40%) used in
fetal B cells but rarely in adult B cells..sup.65, 66 Human adult
precursor and mature B cells mainly seem to use DH2 and DH3 family
segments, as evidenced from sequences of complete VH-DH-JH
rearrangements..sup.68
[0120] Although the exact frequencies of incomplete DH-JH couplings
in different types of mature B-cell malignancies are largely
unknown, it is clear that they will at least be lower than those of
VH-JH joinings. Nevertheless, DH-JH rearrangements might still
represent an important complementary target for PCR-based clonality
assessment. This presumed contribution of DH-JH rearrangements as
PCR target is based on the assumption that incomplete
rearrangements in the IGH locus will not to contain somatic
hypermutations, because transcription starting from the promoters
in the V gene segments does not occur, which is regarded as an
essential prerequisite for somatic hypermutation to take
place..sup.67, 68 Especially in those types of B-lineage
proliferations in which somatic hypermutations are frequent, PCR
analysis of a possible DH-JH recombination product might therefore
be relevant, and sometimes even the only possibility to detect the
B-cell clone.
Primer Design
[0121] Based on the high degree of homology within each DH family,
seven family-specific DH primers were designed (FIG. 4) in
combination with the consensus JH primer that is also used for
detection of VH-JH rearrangements (see Example 1) and t(11;14)
(BCL1-IGH) and t(14;18) (BCL2-IGH) (Examples 8 and 9). Primers were
designed such that cross-annealing to other DH family segments
would be minimal or preferably absent, resulting in distinct
positions for the various family primers relative to the RSS
elements (FIG. 4). The expected PCR product sizes of DH-JH joints
range from 110-130 bp (for DH7-JH joinings) to 395-415 bp (for
DH3-JH rearrangements). Of note, due to the position of the DH7-27
segment close to the segments in the JH region, PCR products of 211
bp (and also 419,1031,1404,1804, and 2420 bp in case of primer
annealing to downstream JH gene segments) will be amplified from
non-rearranged alleles and will be detected as a ladder of germline
bands in virtually every sample.
Results of Initial Testing Phase
[0122] For initial testing of the individual DH primers, high tumor
load precursor B-ALL or T-ALL samples with well-defined clonal
DH-JH rearrangements were used. Under standard PCR conditions using
1.5 mM MgCl.sub.2 and AmpliTaq Gold buffer, all seven primer
combinations appeared to detect the clonal DH-JH targets with
product lengths within the expected size ranges. Cross-annealing of
the DH primers to rearranged gene segments from other DH families
was only very weak or not observed at all. Furthermore, also in
healthy control tonsillar or MNC DNA PCR products of the correct
size ranges were observed. Nonspecific annealing of the primers was
not observed for virtually all primers sets, using non-template
specific control DNA; only in case of the DH2 I JH primer set a
(sometimes faint) 340-350 bp product was observed in HeLa DNA.
Further sequencing revealed that this nonspecific product was due
to false priming of the DH2 primer to a DNA sequence upstream of
the JH4 segment. However, as the size of this nonspecific product
was so different from the sizes of any of the true DH-JH PCR
products, it was decided not to design a new DH2 primer. In fact,
the nonspecific 350 bp band can be employed as an internal marker
for successful DNA amplification and hence the quality of the
template DNA, being hardly or only faintly visible when enough
clonal or polyclonal DH-JH template is available (e.g. in tonsillar
DNA or DNA from particular leukemic samples), but being especially
strong in samples containing low numbers of lymphoid cells with
DH-JH rearrangements.
[0123] Serial dilutions of DNA from the clonal reference samples
into tonsillar DNA generally resulted in sensitivities of 5% or
lower (0.5-1% in case of the DH6-JH rearrangement) using IHD
analysis; sensitivities in GS analysis were generally 1-2 dilution
steps better, i.e. 1% or lower. The clonal DH7-JH target could only
be detected with a sensitivity of .about.10%, which is most
probably caused by primer consumption in PCR amplicons involving
the non-rearranged germline DH7 and JH gene segments.
[0124] Although the initial multiplex strategy, as suggested from
the OLIGO 6.2-assisted primer design, was to divide the various DH
primers over two tubes, it was decided after testing various
multiplex approaches to combine all primers into one multiplex tube
(tube D of IGH clonality assay), except for the DH7 primer, which
was included in a separate tube (tube E of IGH clonality assay).
The reason to exclude the DH7 primer was the complicated germline
pattern, due to easy amplification of alleles with non-rearranged
DH7 segments. Using this two-tube multiplex approach, all clonal
reference samples were still detectable. Under multiplex conditions
the detection limits for these various clonal targets were
logically less optimal as compared to the single assays, ranging
from .about.5% (DH3, DH4, and DH6) to 10% (DH2, and DH5). For the
DH1 clonal reference sample that was available, a sensitivity of
20% was observed; at a later stage the DH1-JH rearrangement of cell
line KCA was found to be detectable down to 10% in the multiplex
assay. As tube E only contains the DH7 primer, the 10% sensitivity
for this tube was the same as mentioned before. The same multiplex
analysis performed with 500 ng instead of 100 ng DNA of the serial
dilutions, resulted in slightly better sensitivities. The use of
serial dilutions in MNC DNA instead of tonsillar DNA did not
clearly affect detection limits of the assays for DH-JH
recombinations. Results of general testing phase Following initial
testing in the three laboratories involved in primer design, the
developed IGH DH-JH multiplex PCR assay was further evaluated using
the 90 Southern blot-defined samples. Every sample was analyzed in
parallel in four laboratories by HD analysis and in five
laboratories by GS analysis; in another two laboratories all
samples were analyzed by both techniques. All together a total of
six HD and seven GS analysis results were obtained per sample per
tube. Despite concordant results (>80% of laboratories with
identical results) in the vast majority of samples, nine showed
inter-laboratory discordancies in tube D. Further analysis revealed
that these could be explained by either the presence of a small
clone with weak clonal products, or to large size products
(.about.390 and larger). In a few cases the products were so large,
that only after sequencing it became clear that they concerned true
but extended DHin -JH rearrangements, either from upstream DH (e.g.
DH6-25-DH1-26-JH in NL-12) or from downstream JH gene segments
(e.g. DH6-25-JH4-JH5 in PT-14). In all three cases (NL-17, mycosis
fungoides; FR-1, B-CLL; FR-5, FCL) in which clonal products were
found using tube E, the results were completely concordant between
laboratories.
[0125] When evaluating results from HD and GS analysis, it appeared
that these were comparable, although in general the number of
laboratories showing identical results was slightly higher upon HD
as compared to GS analysis (FIGS. 4B and C).
[0126] Direct comparison of DH-JH multiplex PCR results with SB
data is virtually impossible, as hybridization with a single probe
(IGHJ6) in the JH region does not allow discrimination between
VH-JH and DH-JH rearrangements. In three samples it was clear that
detection of clonal products of the combined VH-JH and DH-JH assays
did not fit with configuration of the IGH locus in SB analysis.
Remarkably, no clonal DH-JH PCR products were observed in the
pre-follicular B-cell malignancies. In contrast, 11/16 B-CLL
samples and 12/25 (post-)follicular B-cell malignancy samples did
contain clonally rearranged DH-JH PCR products. In three of the
eighteen T-cell malignancy cases clonal DH-JH rearrangements were
seen; these concerned T-LBL (ES-9) and mycosis fungoides (NL-17)
cases with SB-detected IGH rearrangements, and a case of T-NHL/EATL
(PT-4) without SB-detected IGH rearrangements, probably because of
the low tumor load of <15%. All 15 reactive cases only showed
polyclonal DH-JH PCR products, in accordance with SB results. In
category D with difficult diagnoses, three samples (PT-12, GBS-10,
and GBN-8) showed clonal IGH DH-JH PCR products, which was in line
with SB data as well as IGK PCR data in two of three cases; in
another two samples (PT-6 and GBS-9), both T-cell rich B-NHL cases,
clonal DH-JH products were found in addition to clonal IGK and/or
IGL products, but without evidence for clonality from SB analysis,
which might best be explained by the small size of the B-cell clone
in these samples.
[0127] In order to determine the additional value of DH-JH PCR
analysis, the results were compared to those of VH-JH PCR analysis.
In five (NL-4, PT-14, GBN-2, FR-7, NL-12) B-cell malignancies
clonal DH-JH PCR products were found, whereas only polyclonal VH-JH
PCR products were observed.
Conclusion
[0128] In conclusion, based on the initial and general testing
phases, DH-JH PCR analysis appears to be of added value for
clonality assessment. Although HD analysis results might be
interpreted slightly more easily, there is no clear preference for
either HD or GS analysis as they are both suitable for analyzing
amplified PCR products. A potential difficulty in DH-JH PCR
analysis is the relatively large size range of expected PCR
products, due to scattered primer positions and to extended
amplifications from upstream DH or downstream JH gene segments,
implying that long runs are recommended for GS analysis. Finally,
the remarkable position of the DH7-27 gene segment in the IGH locus
causes a ladder of germline amplification products in tube E, with
clonal products being easily recognizable as much smaller
bands/peaks.
EXAMPLE 3
IGK Gene Rearrangements: V.kappa.-J.kappa.,
V.kappa.-Kde/IntronRSS-Kde
Background
[0129] The human IGK light chain locus (on chromosome 2p11.2)
contains many distinct V.kappa. gene segments, grouped into seven
V.kappa. gene families, as well as five J.sub.K gene segments
upstream of the C.kappa. region. Originally, the V.kappa. gene
segments were designated according to the nomenclature as described
by Zachau et al..sup.69 An alternative nomenclature groups the
V.kappa. gene segments in seven families and is used in the
ImMunoGeneTics database..sup.48 ere we follow the latter
nomenclature. The V.kappa.1, V.kappa.2, and V.kappa.3 families are
multi-member families including both functional and pseudo gene
segments, whereas the other families only contain a single
(V.kappa.4, V.kappa.5, V.kappa.7) or a few segments
(V.kappa.6)..sup.70 Remarkably, all V.kappa. gene segments are
dispersed over two large duplicated clusters, one immediately
upstream and in the same orientation as the J.kappa. segments, and
the other more distal and in an inverted orientation (FIG.
5A)..sup.71 The latter implies that so-called inversion
rearrangements are required to form V.kappa.-J.kappa. joints
involving V.kappa. genes of the distal cluster. In addition to the
V.kappa. and J.kappa. segments, there are other elements in the IGK
locus that can be involved in recombination. The kappa deleting
element (Kde), approximately 24 kb downstream of the
J.kappa.-C.kappa. region, can rearrange to V.kappa. gene segments
(V.kappa.-Kde), but also to an isolated RSS in the
J.kappa.-C.kappa. intron (intronRSS-Kde)..sup.24,72 Both types of
rearrangements lead to functional inactivation of the IGK allele,
through deletion of either the C.kappa. exon (intronRSS-Kde
rearrangement) or the entire J.kappa.-C.kappa. area (V.kappa.-Kde
rearrangement).
[0130] As human IGK recombination starts in precursor B-cells in
the bone marrow, IGK rearrangements can also be detected in
precursor B-cell acute leukemias (30-45% of alleles, depending on
age). Although V.kappa.-J.kappa. joinings are present, these IGK
rearrangements mainly concern recombinations involving Kde (25-35%
of alleles). In childhood precursor B-ALL V.kappa.-Kde
recombination predominates over intron-Kde, whereas in adult ALL
the deletions exclusively concern V.kappa.-Kde couplings..sup.24,
73, 74 In chronic B-cell leukemias IGK rearrangements are even more
frequent, being detectable on 75% (Ig.kappa..sup.+ cases) or even
95% (Ig.lamda..sup.+ cases) of all IGK alleles. By definition,
functional V.kappa.-J.kappa. rearrangements are found on at least
one allele in Ig.kappa..sup.+ B-cell leukemias; the non-coding
second allele is either in germline configuration, or inactivated
through V.kappa.-Kde (8% of alleles) or intronRSS-Kde (8% of
alleles) recombination. Kde rearrangements are frequent in
Ig.lamda..sup.+ B-cell leukemias (.about.85% of alleles), with a
slight predominance of intronRSS-Kde recombinations over
V.kappa.-Kde rearrangements. This implies that virtually all
Ig.lamda..sup.+ leukemias contain a Kde rearrangement, while
potentially functional V.kappa.-J.kappa. couplings are relatively
rare..sup.24, 75 Several studies have shown that V.kappa. gene
segment usage is almost identical between various normal and
malignant B-cell populations and largely reflects the number of
available gene segments within each family. Both in
V.kappa.-J.kappa. as well as in V.kappa.-Kde rearrangements,
V.kappa. gene segments from the first four families (V.kappa.1 to
V.kappa.4) predominate. V.kappa.2 gene usage appeared to be higher
in precursor B-ALL than in more mature B-cell lymphoproliferations
or normal B cells. Remarkably, the distal inverted V.kappa. cluster
was rarely used in V.kappa.-J.kappa. rearrangements, whereas
V.kappa. pseudogene segments were never involved, also not in
Ig.lamda..sup.+ cases..sup.76 Little is known about J.kappa. gene
segment usage, but sparse data show that J.kappa.1, J.kappa.2, and
J.kappa.4 are the most frequently used J.kappa. gene
segments..sup.75
[0131] V.kappa.-J.kappa. rearrangements can be important
complementary PCR target for those types of B-cell proliferations
in which somatic hypermutations may hamper amplification of the
VH-JH target, but recombinations involving Kde are probably even
more valuable. Deletion of intervening sequences in the
J.kappa.-C.kappa. intron results in the removal of the IGK
enhancer, which is thought to be essential for the somatic
hypermutation process to occur. Rearrangements involving Kde are
therefore assumed to be free of somatic hypermutations, and hence
should be amplified rather easily.
Primer Design
[0132] Using OLIGO 6.2 software, six family-specific V.kappa.
primers were designed to recognize the various V.kappa. gene
segments of the seven V.kappa. families; the V.kappa.6 family gene
segments were covered by the V.kappa.1 family primer (FIG. 5B). In
case of the relatively large V.kappa.1, V.kappa.2, and V.kappa.3
families only the functional V.kappa. gene segments were taken into
consideration, as the less homologous pseudo gene segments
complicated optimal primer design too much. The family-specific
V.kappa. primers were designed to be used in combination with
either a set of two J.kappa. primers (J.kappa.1-4, covering the
first four J.kappa. segments and J.kappa.5 covering the fifth) or a
Kde primer (FIG. 5B). For analysis of Kde rearrangements an
additional forward primer recognizing a sequence upstream of the
intronRSS was made. In order to show minimal cross-annealing to
other V.kappa. family segments and still be useful in multiplex
reactions, the various primers could not be designed at similar
positions relative to RSS elements (FIG. 5B). The expected PCR
product sizes of V.kappa.-J.kappa. joints range from .about.115-135
bp (for V.kappa.7-J.kappa. joints) to .about.280-300 bp
(V.kappa.2-J.kappa. rearrangements). For the Kde rearrangements,
product size ranges are from .about.195-215 bp (V.kappa.7-Kde) to
.about.360-380 bp (V.kappa.2-Kde), whereas the intronRSS-Kde
products are .about.275-295 bp.
Results of Initial Testing Phase
[0133] For initial testing of the individual primers, several cell
lines and patient samples with precisely defined clonal
V.kappa.-J.kappa., or V.kappa.-Kde/intronRSS-Kde rearrangements
were used. The patient samples with V.kappa.-J.kappa. joints mostly
concerned chronic B-cell leukemias, which were additionally
selected on basis of a high tumor load for easy and sensitive
detection of the involved rearrangement. Unfortunately, clonal
reference samples were not available for all V.kappa.-J.kappa.
targets; especially the more rare types of rearrangements involving
V.kappa.5, V.kappa.7 and/or J.kappa.5 were not represented in the
series of reference samples. For these targets and also for the
targets for which clonal reference samples were available, healthy
control tonsillar or MNC DNA samples were employed, in which PCR
products of the correct expected sizes were indeed observed. The
only exception was the V.kappa.7/J.kappa.5 primer combination; most
probably V.kappa.7-J.kappa.5 joinings are so rare in normal B
cells, that these PCR products were hardly or not detectable in
tonsils. Rearranged products within the expected size ranges could
be detected in all clonal reference samples, under standard PCR
conditions using 1.5 mM MgCl.sub.2 and either ABI Gold Buffer or
ABI Buffer II. However, in a few cases weak amplification of
particular V.kappa.-J.kappa. rearrangements was observed with other
V.kappa. family/J.kappa. primer sets, due to slight cross-annealing
of the V.kappa.3 primer to a few V.kappa.1 gene segments.
Furthermore, in a few of the clonal reference samples clear
additional clonal PCR products were seen with other
V.kappa./J.kappa. or even V.kappa./Kde and intronRSS/Kde primer
sets; in most samples this could be explained by the complete
configuration of the two IGK alleles. This occurrence of multiple
clonal PCR products illustrates the complexity of IGK rearrangement
patterns in a given cell sample, mainly caused by the potential
occurrence of two clonal rearrangements on one allele
(V.kappa.-J.kappa. and intron RSS-Kde). This complexity does not
hamper but support the discrimination between polyclonality and
monoclonality.
[0134] No nonspecific annealing of the primers was observed for any
of the V.kappa.-J.kappa. and V.kappa.-Kde/intron RSS-Kde primer
sets, when using HeLa DNA as a non-template specific control.
Serial dilutions of DNA from the clonal reference samples into
tonsillar DNA generally resulted in sensitivities of 5-10 % for
V.kappa.-J.kappa. rearrangements and 1-10 % for V.kappa.-Kde
rearrangements, using HD analysis. In general, the sensitivities in
GS analysis were approximately one dilution step better. The only
slightly problematic target was the intronRSS-Kde target that could
only be detected down to the 10% serial dilution in the employed
patient sample. This is probably caused by the fact that
intronRSS-Kde rearrangements are abundant in DNA from both
Ig.kappa..sup.+ and Ig.lamda..sup.+ tonsillar B cells, which were
used in the dilution experiments.
[0135] The multiplex strategy that was chosen after testing several
approaches consisted of two different multiplex PCR reaction tubes.
In the V.kappa.-J.kappa. tube (tube A) all V.kappa. primers were
combined with both J.kappa. primers, whereas tube B contained all
V.kappa. primers plus the intronRSS primer in combination with the
Kde reverse primer (FIG. 5B). All beforementioned clonal reference
samples were detectable using this two-tube multiplex approach. Of
note is the observation that in non-clonal tonsil samples a
predominant, seemingly clonal band of .about.150 bp was detected
using the V.kappa.-J.kappa. multiplex tube A analysis. The presence
of this product, which is seen in HD analysis but especially in GS
analysis, can be explained by the limited heterogeneity of
V.kappa.-J.kappa. junctional regions leading to a high frequency of
products of an average size of .about.150 bp. Furthermore, in some
samples a sometimes weak 404 bp nonspecific band was observed in
tube B. Although sensitivities were on average slightly better in
other multiplex approaches in which the V.kappa. primers were
further subdivided over multiple tubes, the feasibility of having
only two tubes to analyze all relevant IGK rearrangements, finally
was the most important argument for choosing the two-tube multiplex
strategy as given in FIG. 5B. Detection limits for the various
clonal targets in the two-tube multiplex approach were .about.10%
for most of the clonal V.kappa.-J.kappa. rearrangements
(V.kappa.1-J.kappa.4, V.kappa.2-J.kappa.4, V-.kappa.3-J.kappa.4)
derived from informative samples with a high tumor load; several of
the V.kappa.-Kde targets were detectable with a still reasonable
sensitivity of .about.10%, but a few other samples containing
V.kappa.2-Kde, V.kappa.5-Kde, and also intronRSS-Kde targets showed
detection limits above 10%. Even the use of 500 ng serially diluted
DNA instead of 100 ng hardly resulted in better sensitivities,
whereas serial dilutions in MNC DNA did not affect the detection
limits either. Nevertheless, detection limits of serial dilutions
of reference DNA in water were all in the order of 0.5-1%, which
shows that the chosen multiplex IGK PCR assay as such is good. It
is important to note that potential clonal cell populations in
lymph nodes or peripheral blood in practice will have to be
detected within a background of polyclonal cells, which can hamper
sensitive clonality detection, especially in samples with a
relatively high background of polyclonal B-cells.
Results of General Testing Phase
[0136] Following initial testing in the four laboratories involved
in primer design, the developed IGK multiplex PCR assay was further
evaluated using 90 Southern blot-defined samples. Every sample was
analyzed in parallel via HD (five laboratories) and GS (two
laboratories) analysis; in another four laboratories all samples
were analyzed by both techniques. Taken together, eight HD and five
GS analysis results were available per sample per tube. In the vast
majority of samples >80% of laboratories produced identical
results, i.e. either clonal bands/peaks or polyclonal smears/curves
in one or both tubes. However, in nine (.about.10%) samples
discordancies were found between laboratories, which remained after
repetitive analysis of these samples. More detailed analysis
revealed that in at least six cases the approximately 150 and 200
bp sizes of the clonal products in tube A could not easily be
discriminated from polyclonal products of roughly the same size.
This is an inherent difficulty in especially V.kappa.-J.kappa.
analysis, which is caused by the relatively limited junctional
heterogeneity of these rearrangements. In two samples the results
from tube B were however so clear in all laboratories with both
techniques that in fact no discrepancy prevailed. In one sample
(ES-8) a large product of around 500 bp appeared to be the reason
for discrepant inter-laboratory results; further sequencing
revealed that amplification starting from the downstream J.kappa.
segment caused production of an extended
V.kappa.1-J.kappa.3-J.kappa.4 PCR product.
[0137] When evaluating results from HD and GS analysis, it appeared
that these were rather comparable, although in general the number
of laboratories showing identical results was slightly higher upon
HD as compared to GS analysis (FIGS. 5C and D). Remarkably, in one
sample (GBS-4) HD analysis revealed a clear product in both tubes,
whereas GS analysis only showed polyclonality. Cloning of the HD
product showed a peculiar V.kappa.3-V.kappa.5 PCR product, which
was not observed in any other sample; the V.kappa.-V.kappa.
configuration of this product explained why it was not detected
with labeled J.kappa. primers in GS analysis.
[0138] Comparison of PCR results with SB data revealed no SB-PCR
discrepancies in the pre-follicular B-cell malignancies and B-CLL
samples; in line with the presence of rearranged IGK bands in SB
analysis, all samples contained clonal IGK PCR products. In
contrast, in the 25 (post-)follicular B-cell malignancy samples
clonal IGK PCR products were missed in four DLCL cases (ES-5,
PT-13, PT-14, FR-7) and one PC leukemia (NL-19) with both
techniques and in another DLCL case (GBS-4, see above) with GS
analysis only. In all cases this was most probably caused by
somatic hypermutation. Interestingly, in one FCL case (NL-4), a
clonal PCR product was found, whereas SB analysis revealed a
germline band in case of the IGK genes and weak clonal bands upon
IGH analysis. In all 18 T-cell malignancy cases and all 15 reactive
cases (category C) polyclonal IGK PCR products were found in
accordance with SB results, except for one peripheral T-NHL case
(FR-10). Next to the clonal TCR and IGK products this sample also
showed clonal IGH and IGL PCR products, but no clonal Ig
rearrangements in SB analysis, probably reflecting the presence of
a small additional B-cell clone in this sample. Finally, in the
category with difficult diagnoses (D), two samples (GBS-10 and
GBN-8) showed clonal IGK PCR products, in line with SB data;
however, in another two samples (PT-6 and GBS-9), both T-cell rich
B-NHL cases, clonal IGK PCR products were found as well as clonal
IGH and/or IGL products, but without evidence for clonality from SB
analysis. Also this discrepancy can probably be explained by the
small size of the B-cell clone in these two patient samples.
[0139] To determine the additional value of analyzing the IGK
locus, we compared the results of IGK PCR analysis to those of IGH
PCR analysis. In five (ES-2, NL-4, PT-8, GBN-2, ES-8) of the nine
samples in which no clonal VH-JH PCR products were found, clonal
products were readily observed in IGK analysis. When taking into
account both VH-JH and DH-JH analysis, IGK PCR analysis was still
complementary to IGH PCR analysis in three of these cases in
detecting clonal Ig PCR products.
Conclusion
[0140] In conclusion, based on the initial and general testing
phases as well as preliminary evidence from use of these multiplex
assays in pathologically well-defined series of
lymphoproliferations, PCR analysis of the IGK locus has clear
(additional) value for clonality detection. Nevertheless, care
should be taken with interpretation of seemingly clonal bands in
especially tube A, due to the inherent restricted IGK junctional
heterogeneity. As this problem is especially apparent in GS
analysis, HD analysis is slightly preferred over GS analysis,
although it should be marked that in some cases GS analysis may
facilitate proper interpretation of results. Another potential
pitfall is the relatively large size range of expected rearranged
IGK products, due to scattered primer positions, and to extended
amplifications from downstream J.kappa. gene segments. This implies
that long runs are recommended for GS analysis. Finally, the
inherent complexity of multiple rearrangements in the IGK locus
(V.kappa.-J.kappa. and Kde rearrangements on the same allele),
together with a low level of cross-annealing of V.kappa. primers,
may occasionally result in patterns with multiple bands or peaks,
resembling oligoclonality. However, with these considerations in
mind, the two-tube IGK multiplex PCR system can be valuable in
PCR-based clonality diagnostics.
EXAMPLE 4
IGL Gene Rearrangements
Background
[0141] IGL gene rearrangements are present in 5 to 10% of
Ig.kappa..sup.+ B-cell malignancies and in all Ig.lamda..sup.+
B-cell malignancies..sup.75 Therefore V.lamda.-J.lamda.
rearrangements potentially represent an attractive extra PCR target
for clonality studies to compensate for false-negative IGH VH-JH
PCR results, mainly caused by somatic mutations. The IGL locus
spans 1 Mb on chromosome 22q11.2..sup.77-79 There are 73-74
V.lamda. genes over 900 kb, among which 30-33 are functional (FIG.
6A). Upon sequence homology, the V.lamda. genes can be grouped in
11 families and three clans. Members of the same family tend to be
clustered on the chromosome. The J.lamda. and C.lamda. genes are
organized in tandem with a J.lamda. segment preceding a C.lamda.
gene. Typically there are 7 J-C.lamda. gene segments, of which
J-C.lamda.1, J-C.lamda.2, J-C.lamda.3, and J-C.lamda.7 are
functional and encode the four Ig.lamda. isotypes FIG. 6A)..sup.80,
81 There is however a polymorphic variation in the number of
J-C.lamda. gene segments, since some individual may carry up to 11
of them, due to an amplification of the C.lamda.2-C.lamda.3
region..sup.82 83
[0142] Several studies have shown that the IGL gene repertoire of
both normal and malignant B cells is biased..sup.48, 49, 84, 85
Thus over 90% of V.lamda. genes used by normal B cells belong to
the V.lamda.1, V.lamda.2 and V.lamda.3 families, which comprise 60%
of the functional genes. Moreover, three genes (2-14, 1-40, 2-8)
account for about half of the expressed repertoire. While normal B
cells use J-C.lamda.1, J-C.lamda.2 and J-C.lamda.3 gene segments in
roughly equivalent proportions, neoplastic B cells tend to use
predominantly J-C.lamda.2 and J-C.lamda.3 gene segments..sup.49 In
both normal and malignant B cells the J-C.lamda.7 is used very
rarely (1%). This latter finding was however challenged by a
single-cell study of normal cells which found that more than half
of the rearrangements employed the J-C.lamda.7 gene
segments..sup.86 In contrast to the mouse, there is some junctional
diversity due to exonuclease activity and N nucleotide addition in
human IGL gene rearrangements..sup.82, 85-87 It is however much
less extensive than that of the IGH locus, and a number of
rearrangements result from the directly coupling of germline
V.lamda. and J.lamda. gene segments. Nevertheless, the IGL locus
might represent an alternative complementary locus to IGH for
B-cell clonality studies.
Primer Design
[0143] Considering the biased V.lamda. repertoire, we chose to
amplify only rearrangements which used the V.lamda.1, V.lamda.2 and
V.lamda.3 gene segments. A single consensus primer recognizing both
V.lamda.1 and V.lamda.2 gene segments, as well as a V.lamda.3
primer, were designed in regions of high homology between members
of the same family (FIG. 6B). Initial experiments showed that they
worked as well in multiplex as separately. In fact, cross annealing
of V.lamda.3 primer hybridizing to some V.lamda.1 or V.lamda.2
genes (or vice versa) could be observed when V.lamda. primers were
used separately; it was not seen however in multiplex PCR.
[0144] A single consensus primer was designed for the J.lamda.1,
J.lamda.2 and J.lamda.3 gene segments and has one mismatch in its
central portion compared to each of the germline sequences. In
preliminary experiments it was found to give rather better results
than a combination of perfectly matched J.lamda.1 and
J.lamda.2-J.lamda.3 primers. Since a single study reported the
frequent usage of the J.lamda.7 gene in normal B cells,.sup.88 we
also designed a J.lamda.7 specific primer. When tested on various
polyclonal B cell samples, we could hardly detect any signal in HD
analysis, in contrast to amplifications performed on the same
samples using the J.lamda.1, J.lamda.2-J.lamda.3 or the J.lamda.
consensus primers. Similarly, we could not detect any rearrangement
with this primer when analyzing a collection of monoclonal B-cell
tumors. Based on these results as well as the other reports in the
literature.sup.49, we concluded that the non-confirmed high
frequency of J.lamda.7 rearrangements (in a single study).sup.88
had been caused by a technical pitfall and consequently, we decided
not to include the J.lamda.7 primer. The PCR assay for the
detection of IGL gene rearrangements in clonality study therefore
consists of a single tube containing three primers (FIG. 6B). This
single tube was expected to detect the vast majority of the
rearrangements.
Results of Initial Testing Phase
[0145] Initial testing on a set of monoclonal and polyclonal
samples showed they could very well be differentiated upon HD
analysis of PCR products on 10% polyacrylamide gel electrophoresis
(FIG. 6C). Clonal IGL rearrangements were seen in the homoduplex
region, with one or sometimes two weaker bands in the heteroduplex
region, while polyclonal rearrangements appeared as a smear in the
heteroduplex region (FIG. 6C). Nonspecific bands were not observed.
It should be noted that because of the limited size of the
junctional region, it is extremely difficult to distinguish
polyclonal from monoclonal rearrangements by running a simple
polyacrylamide gel without performing a heteroduplex formation.
Along this line, analysis of PCR products by GS proved to be less
straightforward (FIG. 6C). While monoclonal rearrangements were
clearly identified, the polyclonal rearrangement pattern had an
oligoclonal aspect due to the limited junctional diversity. The
interpretation was more difficult, particularly to distinguish
polyclonal cases from those with a minor clonal B-cell population
in a background of polyclonal B-cells. We therefore recommend HD
analysis as the method of choice to analyze IGL gene
rearrangements.
[0146] The sensitivity of the assay, performed on several cases,
proved to be about 5% (2.5%-10%) when dilution of tumor DNA was
done in PB-MNC and about 10% (5%-20%) when diluted in lymph node
DNA.
Results of General Testing Phase
[0147] The single-tube IGL PCR assay was evaluated on the series of
90 Southern blot defined lymphoid proliferations. This testing was
done by nine laboratories, four with HD analysis only, one with GS
analysis only, and four using both techniques. Clonal IGL gene
rearrangements were detected in 19 cases. In 15 of them more than
70% concordance was obtained within the nine laboratories. In four
cases less than 70% concordancy was obtained, which could be
explained by minor clonal IGL gene rearrangements in three of them
(ES-12, GB-10, and FR-10). This discordancy in the fourth case
(PT-11) remains unexplained, particularly because no IGL gene
rearrangements were detected by Southern blotting. As concluded
from the initial testing, interpretation of GS analysis was more
difficult than HD analysis, especially in the case of minor clonal
populations. Of these 19 clonal IGL gene cases, 17 were B-cell
proliferations (16 mature and one precursor B-cell). One case
(ES12) corresponded to Hodgkin's disease and another (FR-10) to a
T-NHL. Both had only a minor clonal IGL gene rearrangement, and
FR-10 also displayed a clonal IGK gene rearrangement.
[0148] Comparison with Southern blot data showed some
discrepancies. Six cases with clonal IGL gene rearrangements by PCR
appeared as polyclonal by Southern blot analysis. Three of them
(PT-6, ES-12, FR-10) concerned minor clonal populations which may
have been below the sensitivity level of the Southern blot
technique. In the three other cases (NL-19, ES-1, PT-11) a clonally
rearranged band may have been missed by the fairly complex
rearrangement pattern of the IGL locus on Southern blot..sup.26, 49
Conversely the PCR assay failed to detect clonal rearrangements
which were seen by Southern blot analysis in two cases (GBS-6,
FR-5). However these were follicular lymphomas in which a high
degree of somatic hypermutations may have prevented annealing of
the IGL gene primers.
Conclusion
[0149] In conclusion, a single-tube PCR assay for the detection of
IGL gene rearrangements containing only three primers (FIG. 6B)
allows to detect the vast majority of IGL gene rearrangements
(V.lamda.1, V.lamda.2, and V3 gene rearrangements). Heteroduplex
analysis is the preferred analytic method, though GeneScan analysis
can be used, but maximal caution is recommended to avoid
overinterpretation of clonality due to the limited junctional
diversity.
EXAMPLE 5
TCRB Gene Rearrangements: V.beta.-J.beta., D.beta.-J.beta.
Background
[0150] Molecular analysis of the TCRB genes is an important tool
for assessment of clonality in suspect T-cell proliferations. TCRB
gene rearrangements occur not only in almost all mature T-cell
malignancies but also in about 80% of the CD3 negative T-cell acute
lymphoblastic leukemias (T-ALL) and 95% of the CD3 positive
T-ALL..sup.28 TCRB rearrangements are not restricted to T-lineage
malignancies as about one third of precursor-B-ALL harbor
rearranged TCRB genes..sup.30 Their frequency is much lower (0 to
7%) in mature B cell proliferations..sup.21
[0151] The human TCRB locus is located on the long arm of
chromosome 7, at band 7q34 and spans a region of 685 kb. In
contrast to the TCRG and TCRD loci the V region gene cluster of the
TCRB locus is far more complex (FIG. 7A)..sup.1 It contains about
65 V.beta. gene elements for which two different nomenclatures are
used: the one summarized by Arden et al..sup.50 follows the gene
designation of Wei et al..sup.88 and groups the V.beta. genes into
34 families. The alternative nomenclature proposed by Rowen et
al..sup.51 subdivides 30 V.beta. gene subgroups and was later
adopted by IMGT, the international ImMunoGeneTics database
http://imgt.cines.fr (initiator and coordinator: Marie-Paule
Lefranc, Montpellier, France). [Lefranc, 2003#212;Lefranc, 2003
#219] The largest families, V.beta.5, V.beta.6, V.beta.8 an
V.beta.13 (Arden nomenclature) reach a size of seven, nine, five
and eight members, respectively. Twelve V.beta. families contain
only a single member. In general, the families are clearly
demarcated from each other..sup.50 In this report we follow the
Arden nomenclature..sup.50
[0152] 39-47 of the V.beta. gene elements are qualified as
functional and belong to 23 families. 7-9 of the nonfunctional
V.beta. elements have an open reading frame but contain alterations
in the splice sites, recombination signals and/or regulatory
elements. 10-16 are classified as pseudogenes. In addition, a
cluster of six non-functional orphan V.beta. genes have been
reported that are localized at the short arm of chromosome 9
(9p21).sup.89, 90 They are not detected in transcripts..sup.50,
51
[0153] All but one V.beta. genes are located upstream of two
D.beta.-J.beta.-C.beta. clusters. FIG. 7A illustrates that both
C.beta. gene segments (C.beta.1 and C.beta.2) are preceded by a
D.beta. gene (D.beta.1 and D.beta.2) and a J.beta. cluster which
comprises six (J.beta.1.1 to J.beta.1.6) and seven (J.beta.2.1 to
J.beta.2.7) functional J.beta. segments. J.beta. region loci are
classified into two families according to their genomic
localization, not to sequence similarity..sup.51, 88, 91
[0154] Due to the large germline encoded repertoire, the
combinatorial diversity of TCRB gene rearrangements is extensive
compared to the TCRG and TCRD rearrangements. The primary
repertoire of the TCR.beta. molecules is further extended by an
addition of an average of 3.6 (V-D junction) and 4.6 (D-J junction)
nucleotides and deletion of an average of 3.6 (V), 3.8 (5' of D),
3.7 (3' of D) and 4.1 (J) nucleotides..sup.51 The complete
hypervariable region resulting from the junction of the V, D and J
segments comprises characteristically nine or ten codons. Size
variation is limited, as 7 to 12 residues account for more than 80%
of all functional rearrangements in contrast to the broad length
repertoire of the IGH CDR3 region..sup.92
[0155] During early T-cell development the rearrangement of the
TCRB gene consists of two consecutive steps: D.beta. to J.beta.
rearrangement and V.beta. to D-J.beta. rearrangement with an
interval of one to two days between these two processes..sup.93 The
D.beta.1 gene segment may join either J.beta.1 or J.beta.2 gene
segments but the D.beta.2 gene segment generally joins only
J.beta.2 gene segments because of its position in the TCRB gene
locus..sup.28, 51 Due to the presence of two consecutive TCRB D-J
clusters, it is also possible that two rearrangements are
detectable on one allele: an incomplete TCRB D.beta.2-J.beta.2
rearrangement in addition to a complete or incomplete rearrangement
in the TCRB D.beta.1-J.beta.1 region..sup.1
[0156] In TCRB gene rearrangements, a non-random distribution of
gene segment usage is seen. In healthy individuals, some V.beta.
families predominate in the peripheral blood T-cell repertoire (e.g
V.beta.1-V.beta.5), while others are only rarely used (e.g.
V.beta.11, V.beta.16, V.beta.18, V.beta.23). Mean values of the
V.beta. repertoire seem to be stable during aging, although the
standard deviation increase in the elderly..sup.13, 94 Also in the
human thymus some V.beta. gene segments dominate: the most
prevalent seven V.beta. genes (V.beta.3-1, V.beta.4-1, V.beta.5-1,
V.beta.6-7, V.beta.7-2, V.beta.8-2, V.beta.13-2) cover nearly half
of the entire functional TCRB repertoire..sup.85 The representation
of J segments is also far from even. The J.beta.2 family is used
more frequently than the J.beta.1 family (72% vs. 28% of TCRB
rearrangements)..sup.98 In particular, the proportion of J.beta.2.1
is higher than expected (24%) followed by J.beta.2.2 (11%) and
J.beta.2.3 and J.beta.2.5 (10% each)..sup.95
[0157] TCRB gene rearrangement patterns differ between categories
of T cell malignancies. Complete TCRB V.beta.-J.beta.1
rearrangements and incompletely rearranged alleles in the TCRB
D.beta.-J.beta.2 cluster are seen more frequently in
TCR.alpha..beta..sup.+ T-ALL as compared to CD3.sup.- T-ALL and
TCR.gamma..delta..sup.+ T-ALL..sup.28 In the total group of T-ALL
the TCRB D.beta.-J.beta.1 region is relatively frequently involved
in rearrangements in contrast to cross-lineage TCRB gene
rearrangements in precursor-B-ALL which exclusively involve the
TCRB D.beta.-J.beta.2 region..sup.30, 73
[0158] The development of monoclonal antibodies against most
V.beta. domains has helped to identify V.beta. family
expansions..sup.13 However, TCR gene rearrangement analysis is
essential for clonality assessment in T cell lymphoproliferative
disorders. As the restricted germline encoded repertoire of the
TCRG and TCRD loci facilitates DNA based PCR approaches, various
PCR methods have been established for the detection of TCRG and
TCRD gene rearrangements..sup.97-99 Nevertheless, the limited
junctional diversity of TCRG rearrangements leads to a high
background amplification of similar rearrangements in normal T
cells (Example 6). The TCRD gene on the other hand is deleted in
most mature T cell malignancies..sup.21 Therefore DNA based TCRB
PCR techniques are needed for clonality assessment. In addition,
TCRB rearrangements are of great interest for follow-up studies of
lymphoproliferative disorders, because the extensive combinatorial
repertoire of TCRB rearrangements and the large hypervariable
region enables a highly specific detection of clinically occult
residual tumor cells. However, the extensive germline encoded
repertoire renders PCR assays more difficult. Some published PCR
approaches use the time consuming procedure of multiple tube
approaches with a panel of family- or subfamily-specific
primers..sup.96, 100 Usage of highly degenerated consensus primers
limits the number of detectable rearrangements that are
theoretically covered by the primers because there is no single
common sequence of sufficient identity to allow a reliable
amplification of all possible rearrangements..sup.42, 101, 102 Some
published assays use a nested PCR requiring an additional PCR
reaction..sup.42, 102 Other assays focus on analysis of the TCRB
V.beta.-D.beta.-J.beta.-C.beta. transcripts to limit the number of
primers needed..sup.16, 100, 103 However, a major drawback of these
mRNA based approaches is the need for fresh or frozen material and
a reverse transcription step before the PCR amplification.
[0159] We tried to overcome these limitations by creating a
completely new and convenient DNA based TCRB PCR. We designed
multiple V.beta. and J.beta. primers, covering all functional
V.beta. and J.beta. gene segments and being suitable for
combination in multiplex PCR reactions. In addition the assay is
applicable for HD and GS analysis and also detects the incomplete
TCRB D.beta.-J.beta. rearrangements with the same set of J.beta.
primers. In order to avoid problems due to cross priming we decided
to design all V.beta. and J.beta. primers at the same conserved
region of each gene segment.
Primer Design
[0160] Initially a total of 23 V.beta., 2 D.beta. (D.beta.1 and
D.beta.2) and 13 J.beta. (D.beta.1.1 to 1.6 and J.beta.2.1 to 2.7)
primers were designed with all the V.beta. and J.beta. primers
positioned in the same conserved region of each V.beta. and J.beta.
gene segment so that the effects of cross-annealing in a multiplex
reaction could be neglected. In addition, rare polyclonal TCRB V-J
rearrangements would not be mistaken for a clonal rearrangement
even if they do not produce a fully polyclonal Gaussian peak
pattern, because PCR products of all possible rearrangements are
situated in the same size range.
[0161] For primer design, the rearrangeable pseudogenes or open
reading frame genes with alterations in splicing sites,
recombination signals and/or regulatory elements or changes of
conserved amino acids were taken into consideration whenever
possible. However, the main objective was to cover all functional
V.beta. genes. The priming efficiency of each V.beta. primer was
checked for every V.beta. gene using OLIGO 6.2 software. This led
to primers that were not strictly V.beta. family specific and some
of which covered V.beta. gene segments of more than one family
(FIG. 7B). Since the 13 J.beta. primers annealed to the same
segment of each J.beta. gene primer, dimerization made it necessary
to split the J primers into two tubes. Initially, it was planned to
use the primers in four sets of multiplex reactions as follows: all
23 V.beta. primers in combination with the six J.beta.1 family
primers (240-285 bp), all 23 V.beta. primers with the seven
J.beta.2 family primers (240-285 bp), D.beta.1 (280-320 bp) with
the six J.beta.1 primers, and D.beta.1 (280-320 bp) plus D.beta.2
(170-210 bp) with the seven J.beta.2 family primers.
Results of Initial Testing Phase
[0162] Initial monoplex testing of each possible primer combination
was done using samples with known monoclonal TCRB rearrangements
and polyclonal controls. PCR products of the expected size range
were generated with differences in product intensity and signal
profile for polyclonal samples depending on the frequency of usage
of distinct V.beta. and J.beta. gene segments. However, when the
primers were combined in a multiplex reaction some J.beta.2
rearrangements in particular were missed and nonspecific products
in the tubes B and D were observed. In addition cross-priming
between the J.beta.1 and J.beta.2 primers resulted in
interpretation problems. As a consequence the J.beta.2 primers had
to be redesigned and the primer combinations in the different tubes
had to be rearranged: J.beta. primers J.beta.2.2, 2.6 and 2.7 were
slightly modified and added to tube A. The localization of the
primers J.beta.2.1, 2.3, 2.4 and 2.5 was shifted by 4 bp downstream
to avoid primer dimerization and cross priming with the remaining
J.beta. primers. Only nonspecific bands with varying intensity
outside the expected size range persisted in tube B (bands <150
bp, 221 bp) and tube C (128 bp, 337 bp) using specific template
DNA. However, because all nonspecific amplification products were
outside the size ranges of the TCRB specific products, they did not
affect interpretation and were considered not to be a problem.
However, using nonspecific template controls one additional faint
273 bp aspecific peak in tube A was visible by GS analysis. This
product is completely suppressed when the DNA contains enough
clonal or polyclonal TCRB rearrangements but can appear in samples
comprising low numbers of lymphoid cells. In the initial testing
phase relatively faint V-D-J PCR products were generated. Thus we
optimized PCR conditions for complete V-D-J rearrangements by
increasing MgCl.sub.2 concentration and the amount of Taq
polymerase. Also usage of highly purified primers and application
of ABI Buffer II instead of ABI Gold Buffer turned out to be very
important. For detection of the incomplete D.beta.-J.beta.
rearrangements, it was finally possible to mix all J.beta. primers
into one tube without loss of sensitivity or information.
Consequently, the total number of multiplex reactions could be
reduced to three tubes.
[0163] The finally approved primer set is (FIG. 7B):
[0164] tube A: 23 V.beta. primers and 9 J.beta. primers:
J.beta.1.1-1.6, 2.2, 2.6 and 2.7
[0165] tube B: 23 V.beta. primers and 4 J.beta. primers:
J.beta.2.1, 2.3, 2.4 and 2.5
[0166] tube C: D.beta.1, D.beta.2 and all 13 J.beta. primers.
[0167] As tubes A and C contain J.beta.1 and J.beta.2 primers,
differential labeling of J.beta.1 and J.beta.2 primers with
different dyes (TET for J.beta.1.1-1.6 and FAM for J.beta.2.1-2.7
primers) allows GS discrimination of J.beta.1 or J.beta.2 usage in
tube A and C reactions (see FIG. 13A).
[0168] Sensitivity testing was performed via dilution experiments
with various cell lines and patient samples with clonally
rearranged TCRB genes in MNC. Single PCR dilution experiments
generally reached sensitivity levels of at least 0.1% to 1%. As
expected, the sensitivity decreased in multiplex testing, probably
due to an increase of background amplification. Especially in GS
analysis this background hampered interpretation due to the
relative small length variation of the TCRB PCR products.
Nevertheless, in 40 of 46 positive controls tested a sensitivity of
at least 1% to 10% was reached using heteroduplex or GeneScanning
(Table 6).
Results of General Testing Phase
[0169] Eleven groups participated in the analysis of DNA from a
series of 90 Southern blot-defined malignant and reactive
lymphoproliferative disorders using the TCRB multiplex protocol.
Every sample was analysed by HD in two laboratories and in six
laboratories using GS analysis. Another three laboratories used
both techniques for PCR analysis (FIGS. 7C, D, and E). This testing
phase as well as experience from use of these TCRB PCR assays
raised some general issues about the protocol that were in part
already described in the initial testing phase: 1. The limited
length variation of the TCRB PCR products may hamper GS detection
of clonal signals within a polyclonal background. 2. Only
bands/peaks within the expected size range represent clonal TCRB
gene rearrangements. Especially for tube A a nonspecific control
DNA must be included to define the aspecific 273 bp peak that may
occur in situations without competition. 3. It is extremely
important to use highly purified primers and ABI Buffer II (and not
ABI Gold Buffer) for good PCR results as well as the recommended
PCR product preparation protocol for HD analysis. Of the 90
Southern blot-defined cases submitted, 29 were SB positive for
monoclonal TCRB rearrangements. 25 of these clonal rearrangements
(86%) were also detectable by the TCRB PCR. 23 rearrangements were
disclosed by GS and HD analysis, two additional cases only by HD.
One of the GS negative HD positive cases (FR-9) was interpreted as
monoclonal on GS analysis by four of the nine laboratories involved
in the general testing phase (FIG. 7C). However, due to a
significant polyclonal background, interpretation of the GS
patterns was difficult in this particular case. The other GS
negative HD positive case displayed an atypical PCR product in tube
C with a size of about 400 bp (FIG. 7E). The PCR product was
clearly visible in agarose gels and HD analysis but not by GS.
After DNA sequencing of this fragment a TCRB D.beta.1-D.beta.2
amplification product was identified explaining the unlabelled PCR
product. Four SB positive cases (NL-15, NL-16, GBN-2 and FR-6) were
neither detected by GS nor by HD analysis all of them with an
underlying B lymphoid malignancy. Possible explanations for this
failure are atypical rearrangements (e.g. incomplete
V.beta.-D.beta. rearrangements),.sup.28, 104 sequence variations of
the rearranged V.beta. gene segments.sup.51 or a lack of
sensitivity for particular rearrangements.
[0170] 62 of the samples were considered to be polyclonal by SB.
For 61 (98%) of these cases PCR results were concordant with at
least one method of analysis, for 57 (92%) cases results were
concordant using both methods. The one SB negative sample (ES-14)
found to be monoclonal by HD and GS analysis showed an incomplete
D.beta.2-rearrangement. For four samples non-uniform results were
obtained: one sample was considered to be clonal by GS but only by
50% of the labs analyzing the PCR products by HD (GBS-4). Three
samples were found to produce weak clonal signals only by HD
analysis (ES-6, GBS-9 and DE-2). TCRB rearranged subclones being
too small to be detected by SB analysis may only be seen by the
more sensitive PCR methodology. In B cell malignancies the detected
rearrangements may also represent clonal or oligoclonal expansions
of residual T cells..sup.105 In this case these weak clonal PCR
products should not be regarded as evidence of a clonal T cell
disorder. This stresses the importance of the interpretation of the
PCR results in context with other diagnostic tests and the clinical
picture of the patients. Optimal PCR assessment of TCRB
rearrangements is obtained by the combined use of HD and GS
analysis. Sensitivity may differ between the two detection methods
as a function of clonal PCR product size compared to the polyclonal
size distribution: on the one hand HD analysis disperses the
polyclonal background from the clonal products and on the other
hand PCR products outside the main size range allow a more
sensitive GS detection. Also the risk of false-positive results is
reduced in the combined use of HD and GS analysis. Furthermore, HD
analysis allows detection of some additional atypical TCRB D.beta.
1-D.beta.2 rearrangements that cannot be detected by GS analysis of
the PCR product as no labeled primer is involved in amplification.
However, GS analysis is in general the more informative method for
samples with a high tumor load because the exact size of the
monoclonal PCR product is indicated, which may be used for
monitoring purposes and differentially labeled J.beta. primers
provide additional information about J.beta. gene usage.
Conclusion
[0171] In conclusion, the three-tube TCRB multiplex PCR system
provides a new and convenient assay for clonality assessment in
suspect T-cell proliferations with an unprecedentedly high
clonality detection rate.
EXAMPLE 6
TCRG Gene Rearrangements
Background
[0172] TCRG gene rearrangements have long been used for DNA PCR
detection of lymphoid clonality and represent the "prototype" of
restricted repertoire targets. It is a preferential target for
clonality analyses since it is rearranged at an early stage of T
lymphoid development, probably just after TCRD,.sup.106 in both
TCR.alpha..beta. and TCR.gamma..delta. lineage precursors. It is
rearranged in greater than 90% of T-ALL, T-LGL and T-PLL, in 50-75%
of peripheral T-NHL and mycosis fungdides but not in true NK cell
proliferations. It is also rearranged in a major part of B lineage
ALLs, but much less so in B-NHL..sup.1, 30, 73 Unlike several other
Ig/TCR loci, the complete genomic structure has been known for many
years. It contains a limited number of V.gamma. and J.gamma.
segments. Amplification of all major V.gamma.-J.gamma. combinations
is possible with limited number of four V.gamma. and three J.gamma.
primers.
[0173] The human TCRG locus on chromosome 7p14 contains 14 V.gamma.
segments, only ten of which have been shown to undergo
rearrangement (FIG. 8A). The expressed V.gamma. repertoire includes
only six V.gamma. genes (V.gamma.2, V.gamma.3, V.gamma.4,
V.gamma.5, V.gamma.8 and V.gamma.9) but rearrangement also occurs
with the .psi.V.gamma.7, .psi.V.gamma.10, .gamma..psi.V.gamma.11
segments..sup.107, 108 Rearrangement of .psi.V.gamma.B (also known
as V.gamma.12).sup.107 is so exceptional that it is rarely used in
diagnostic PCR strategies. Rearranging V.gamma. segments can be
subdivided into those belonging to the V.gamma.I family
(V.gamma.fI: V.gamma.2, V.gamma.3, V.gamma.4, V.gamma.5,
.psi.V.gamma.7 and V.gamma.8; overall homology >90% and highest
between V.gamma.2 and V.gamma.4 and between V.gamma.3 and
V.gamma.5) and the single member V.gamma.9, .psi.V.gamma.10,
.psi.V.gamma.11 families. The TCRG locus contains five J.gamma.
segments: J.gamma.1.1 (J.gamma.P1), J.gamma.1.2 (J.gamma.P),
J.gamma.1.3 (J.gamma.1), J.gamma.2.1 (J.gamma.P2), J.gamma.2.3
(J.gamma.2), of which J.gamma.1.3 and J.gamma.2.3 are highly
homologous, as are J.gamma.1.1 and J.gamma.2.1..sup.109
[0174] Whilst the restricted TCRG germline repertoire facilitates
PCR amplification, the limited junctional diversity of TCRG
rearrangements complicates distinction between clonal and
polyclonal PCR products. The TCRG locus does not contain D segments
and demonstrates relatively limited nucleotide additions. TCRG V-J
junctional length therefore varies by 20-30 bp, compared to
approximately 60 bp for IGH and TCRD. The capacity to distinguish
clonal from polyclonal TCRG rearrangements depends on the
complexity of the polyclonal repertoire. In general, minor clonal
populations using frequent V.gamma.-J.gamma. rearrangements such as
V.gamma.fI-J.gamma.1.3/2.3 are at risk of being lost amidst the
polyclonal repertoire, whereas rare combinations will be detected
with greater sensitivity. However, occasional polyclonal T
lymphocytes demonstrating rare V.gamma.-J.gamma. rearrangements may
be mistaken for a clonal rearrangement, due to absence of a
polyclonal background for that type of rearrangement. A further
possible source of false positivity results from the presence of
TCR.gamma..delta. expressing T lymphocytes demonstrating
"canonical" TCRG rearrangements, which do not demonstrate N
nucleotide additions. The most commonly recognized human canonical
TCRG rearrangement involves the V.gamma.9-J.gamma.1.2 segments and
occurs in approximately 1% of blood T-lymphocytes..sup.110 111 It
is therefore extremely important to analyze TCRG PCR products using
high resolution electrophoretic techniques or to separate PCR
products on criteria other than purely on size, in order to reduce
the risk of false positive results. It is also important to be
aware of the profile of canonical rearrangements and the situations
in which they most commonly occur. Canonical V.gamma.9-J.gamma.1.2
rearrangements, for example, are found predominantly in peripheral
blood and increase in frequency with age, since they result from
accumulation of TCR.gamma..delta..sup.+ T-lymphocytes..sup.19
[0175] Unlike TCRD, TCRG is not deleted in TCR.alpha..beta. lineage
cells. Since TCRG rearrangements occur in both TCR.alpha..beta. and
TCR.gamma..delta. lineage precursors, their identification cannot
be used for determination of the type of T cell lineage. Similarly,
TCRG rearrangements occur in 60% of B lineage ALLs,.sup.30 implying
that they can not be used for assessment of B vs. T cell lineage in
immature proliferations. However, they occur much less frequently
in mature B lymphoproliferative disorders, including the majority
of B-NHL,.sup.1 and might therefore be used, in combination with
clinical and immunophenotypic data, to determine lineage
involvement in mature lymphoproliferative disorders.
[0176] The limited germline repertoire allows determination of
V.gamma. and J.gamma. segment utilization, either by Southern blot
or PCR analysis. Identification of V.gamma. and J.gamma. usage is
not of purely academic interest, since specific amplification is
required for MRD analysis..sup.112
[0177] We undertook to develop a minimal number of multiplex TCRG
strategies which would maintain optimal sensitivity and
informativity, minimize the risk of false positive results and
allow simple V.gamma. and J.gamma. identification, including by HD
analysis or monofluorescent GS strategies. We chose to include
V.gamma. primers detecting all rearranging segments other than
.psi.V.gamma.B (.psi.V.gamma.12), given its rarity. In order to
reduce the risk of falsely identifying canonical rearrangements as
clonal products, we excluded the J.gamma.1.2 primer, since it is
rarely involved in lymphoid neoplasms and is usually, although not
always, associated with a TCRG rearrangement on the other
allele..sup.113
Primer Design
[0178] We initially developed 3V.gamma. and 2J.gamma. primers, to
be used in two multiplex reactions, as follows: one tube with
J.gamma.1.3/2.3 with V.gamma.9 specific (160-190 bp), V.gamma.fI
consensus (200-230 bp) and V.gamma.10/11 consensus (220-250 bp) and
a second tube with J.gamma.1.1/2.1 with V.gamma.9 specific (190-220
bp), V.gamma.fI consensus (230-260 bp) and V.gamma.10/11 consensus
(250-280bp). V.gamma. usage was to be identified by PCR product
size by HD analysis. No distinction between J.gamma.1.3 and
J.gamma.2.3 or J.gamma.1.1 and J.gamma.2.1 was attempted.
Results of Initial Testing Phase
[0179] While all V.gamma.-J.gamma. combinations gave the expected
profiles on single PCR amplification, multiplex amplification led
to competition of larger PCR products, with preferential
amplification of smaller fragments, and failure to detect some
V.gamma.fI and V.gamma.10/11 rearrangements. This was further
complicated by significant primer dimer formation between the
V.gamma.10/11 consensus and the V.gamma.fI primers. Competition
between differently sized fragments and primer dimer formation both
led to unsatisfactory sensitivity and informativity and this
strategy was therefore abandoned.
[0180] We reasoned that competition would be minimized by
separating the most frequently used V.gamma. primers (V.gamma.fI
and V.gamma.9) and combining them with V.gamma.10 and V.gamma.11
specific primers, respectively. The latter rearrangements are
rarely used and therefore minimize competition for the predominant
repertoires. The V.gamma.10/11 consensus primer was therefore
replaced by two specific V.gamma. primers which generated smaller
PCR products (FIG. 8B). By mixing J.gamma.1.3/2.3 and
J.gamma.1.1/2.1 it was possible to maintain a two-tube multiplex
which allows approximate identification on the basis of product
size of V.gamma. usage by HD analysis and of both J.gamma. and
V.gamma. usage by GS analysis.
[0181] The approved set of multiplex TCRG PCR tubes with four
V.gamma. and two J.gamma. primers includes (FIG. 8B):
[0182] Tube A:
V.gamma.fI+V.gamma.10+J.gamma.1.1/2.1+J.gamma.1.3/2.3
[0183] Tube B:
V.gamma.9+V.gamma.11+J.gamma.1.1/2.1+J.gamma.1.3/2.3
[0184] The position and the sequence of the primers are shown in
FIG. 8B. These primers gave satisfactory amplification in both
single and multiplex PCR formats and allowed detection of virtually
all known V.gamma.-J.gamma. combinations. The competition of larger
PCR fragments was no longer seen, although it cannot be excluded
that some competition of V.gamma.9 or V.gamma.fI rearrangements may
occur if these are present in a minority population. Sensitivity of
detection varied between 1% and 10%, as a function of the
complexity of the polyclonal repertoire and the position of the
clonal rearrangement relative to the polyclonal Gaussian
peak..sup.114 Interpretation of .psi.V.gamma.11 rearrangements can
be difficult, since the normal repertoire is extremely restricted
and since these primitive rearrangements are often present in
subclones.
[0185] Since the V.gamma.4 segment is approximately 40 bp longer
than the other V.gamma.fI members and V.gamma.4 rearrangements are
relatively common in both physiological and pathological lymphoid
cells, the polyclonal repertoire can be skewed towards larger sized
fragments, and clonal V.gamma.4-J.gamma.1.3/2.3 rearrangements
could theoretically be mistaken for V.gamma.fI-J.gamma.1.1/2.1
rearrangements. The proximity of the different repertoires also
makes V.gamma. and J.gamma. identification much more reliable if
differently labeled J.gamma. primers are used. For example, the use
of a TET-labeled J.gamma.1.1/2.1 and a FAM labeled J.gamma.1.3/2.3
was tested in a single center and was shown to give satisfactory
results (FIG. 13B). It is, however, possible to estimate V.gamma.
and J.gamma. usage following GS analysis on the basis of size alone
(FIGS. 8C and D).
Results of General Testing Phase
[0186] Given the limited germline TCRG repertoire and the
restricted junctional diversity, reactive T lymphocytes which have
undergone TCRG rearrangements using a single V.gamma. and J.gamma.
segment with variable CDR3 sequences which are of uniform length,
will migrate as an apparent clonal population by GS analysis. HD
formation will disperse these rearrangements more easily and will
therefore prevent their erroneous interpretation as evidence of
lymphoid clonality. In contrast, GS analysis provides improved
resolution and sensitivity compared to HD analysis: For these
reasons, optimal assessment of TCRG rearrangements requires both HD
and GS analysis. If this is not possible, HD analysis alone is
probably preferable, since it might be associated with a risk of
false-negative results, whereas GS analysis alone will increase the
risk of false-positive results.
[0187] Of the 18 TCRG rearrangements detected by Southern blotting
in the 90 cases, 16 were also detected by PCR. The minor
V.gamma.fI-J.gamma.1.3/2.3 rearrangement detected by Southern in
the NL-1 oligoclonal case, was only detected by PCR in a proportion
of laboratories performing GS analysis. A major
V.gamma.9-J.gamma.1.3/2.3 rearrangement detected in GBS-6 was found
to be polyclonal by both HD and GS in all laboratories and, as
such, probably represents a false-negative result.
[0188] Comparison of allele identification showed that, for all
alleles identified by Southern blotting, PCR V.gamma. and J.gamma.
identification on the basis of size gave concordant results. Seven
rearrangements were detected by Southern blotting but precise
allele identification was not possible. Six of these were due to
J.gamma.1.1/2.1 usage, suggesting that PCR allows preferential
detection of this type of rearrangement.
[0189] Seventy two samples were considered to be polyclonal by
Southern. Sixteen (22%) of these demonstrated a total of 24
rearrangements by TCRG PCR. Of these, 13 (81%) were B lymphoid
proliferations. Sixteen of the 24 clonal rearrangements were minor,
with 15 only being detected by GS in the majority of laboratories.
It is worth noting that, of these minor rearrangements, nine (39%)
involved the .psi.V.gamma.10 segment and eight (33%) V.gamma.9.
.psi.V.gamma.11 rearrangements were not detected. No
.psi.V.gamma.10 rearrangements were detected by Southern blot
analysis. PCR therefore allowed more sensitive detection of minor
clonal .psi.V.gamma.10 rearrangements, particularly by GS analysis.
It is likely that these rearrangements represent residual,
predominantly TCR.alpha..beta. lineage, T lymphocytes with a
restricted repertoire, which may or may not be related to the
underlying B lymphoid malignancy. These minor peaks should
obviously not be interpreted as evidence of a clonal T cell
disorder. They emphasize the importance of understanding the nature
of TCRG rearrangements before using this locus as a PCR target in
the lymphoid clonality diagnostic setting. Consequently, it is also
extremely important to interpret TCRG gene results within their
clinical context.
Conclusion
[0190] In conclusion, the two TCRG multiplex tubes allow detection
of the vast majority of clonal TCRG rearrangements. The potential
risk of false positive results due to over-interpretation of minor
clonal peaks can be minimized by the combined use of heteroduplex
analysis and GeneScanning and by interpreting results within their
clinical context, particularly when the apparent clonality involved
the .psi.V.gamma.10 and .psi.V.gamma.11 segments. The relative
merits of TCRG compared to TCRB analysis for the detection of
clonal T lymphoproliferative disorders should be studied
prospectively. They are likely to represent complementary
strategies.
EXAMPLE 7
TCRD Gene Rearrangements: V.delta.-D.delta.-J.delta.,
D.delta.-D.delta., V.delta.-D.delta., and D.delta.-J.delta.
Background
[0191] The human TCRD gene locus is located on chromosome 14q11.2
between the V.alpha. and J.alpha. gene segments. The major part of
the TCRD locus (D.delta.-J.delta.-C.delta.) is flanked by
TCRD-deleting elements, .psi.J.alpha. and .delta.REC such that
rearrangement of the deleting elements to each other or
rearrangement of V.alpha. to J.alpha. gene segments causes deletion
of the intermediate TCRD gene locus (FIG. 9A). The germline encoded
TCRD locus consists of 8V.delta., 4J.delta., and 3D.delta. gene
segments, of which at least five of the eight V.delta. gene
segments can also rearrange to J.alpha. gene segments..sup.115
Other V.alpha. gene segments may also be utilized in TCRD gene
rearrangements in rare cases. The WHO-IUIS nomenclature.sup.116 for
TCR gene segments uses a different numbering system for those V
genes used mainly or exclusively in TCR.delta. chains from those
which can be used in either TCR.alpha. or TCR.delta. chains. Thus
TCRDV101S1 (V.delta.1), TCRDV102S1 (V.delta.2) and TCRDV103S1
(V.delta.3) are used almost exclusively in TCRD rearrangements,
whereas TCRADV6S1 (V.delta.4), TCRADV21S1 (V.delta.5) and
TCRADV17S1 (V.delta.6) can be used in either TCR.delta. or .alpha.
chains. TCRADV28S1 (V.delta.7) and TCRADV14S1 (V.delta.8) are used
extremely rarely in TCRD rearrangements.
[0192] The germline-encoded repertoire of the
TCR.gamma..delta..sup.+ T cells is small compared to the
TCR.alpha..beta..sup.+ T cells and the combinatorial repertoire is
even more limited due to preferential recombination in peripheral
blood and thymocyte TCR.gamma..delta..sup.+ T cells. At birth, the
repertoire of cord blood TCR.gamma..delta..sup.+ T cells is broad,
with no apparent restriction or preferred expression of particular
V.gamma./V.delta. combinations. During childhood, however, the
peripheral blood TCR.gamma..delta..sup.+ T cell repertoire is
strikingly shaped so that V.gamma.9/V.delta.2 cells clearly
dominate in adults..sup.117 Studies have shown that V.delta.1 and
V.delta.2 repertoires become restricted with age leading to the
appearance of oligoclonal V.delta.1.sup.+ and V.delta.2.sup.+ cells
in blood and intestine..sup.118 TCR.gamma..delta..sup.+ T cells are
evenly distributed throughout human lymphoid tissues but there is
preferential expression of particular V.delta. segments in
specified anatomical localizations. Notably, most intraepithelial
TCR.gamma..delta. T cells occurring in the small intestine and in
the colon express V.delta.1. Similarly, V.delta.1 is expressed by
normal spleen TCR.gamma..delta..sup.+ T cells, but
TCR.gamma..delta..sup.+ T cells in the skin express the V.delta.2
gene.
[0193] Although the small number of V, D and J gene segments
available for recombination limits the potential combinatorial
diversity, the CDR3 or junctional diversity is extensive due to the
addition of N regions, P regions and random deletion of nucleotides
by recombinases. This diversity is also extended by the
recombination of up to three D.delta. segments and therefore up to
four N-regions within the rearranged TCRD locus. This limited
germline diversity encoded at the TCRD locus in conjunction with
extensive junctional diversity results in a useful target for PCR
analysis and TCRD recombination events have been used most
extensively as clonal markers in both T and B cell acute
lymphoblastic leukemia (ALL)..sup.119, 120 The TCRD locus is the
first of all TCR loci to rearrange during T cell ontogeny. The
first event is a D.delta.2-D.delta.3 rearrangement, followed by a
V.delta.2-(D.delta.1-D.delta.2)-D.delta.3 rearrangement, and
finally V.delta.-D.beta.-J.delta. rearrangement. Immature
rearrangements (V.delta.2-D.delta.3 or D.delta.2-D.delta.3) occur
in 70% of precursor B-ALL (and are therefore non lineage
restricted).sup.30 while there is a predominance of mature
rearrangements comprising incomplete D.delta.2-J.delta.1 and
complete V.delta.1, V.delta.2, V.delta.3 to J.delta.1 found in
T-ALL..sup.23, 121 Thus specific primer sets can be used to
identify different types of complete and incomplete rearrangements
corresponding to different types of ALL..sup.122
[0194] TCR.gamma..delta..sup.+ T-ALL form a relatively small
subgroup of ALL, representing 10-15% of T-ALL but still only
constitute 2% of all ALL. V.delta.1-J.delta.1 rearrangements
predominate in TCR.gamma..delta..sup.+ T ALL; interestingly
V.delta.1 is never found in combination with J.delta. segments
other than J.delta.1..sup.15, 20 Other recombinations occur in less
than 25% of alleles. Furthermore, V.delta.1-J.delta.1-C.delta.
chains are almost always disulfide linked to either V.gamma.I or
V.gamma.II gene families recombined to J.gamma.2.3-C.gamma.2. Such
gene usage is consistent with the immature thymic origin of these
leukemic cells.
[0195] Most T cell lymphomas express TCR.alpha..beta. while the
minority express TCR.gamma..delta. and comprise of several distinct
entities. Peripheral T cell lymphomas (PTCL) expressing
TCR.gamma..delta. comprise 8-13% of all PTCL and
V.delta.1-J.delta.1 as well as other V.delta. to J.delta.1
recombinations have been documented..sup.123, 124 Hepatosplenic
.gamma..delta. T-cell lymphoma is derived from splenic
TCR.gamma..delta. T cells which normally express V.delta.1. It is
an uncommon entity that exhibits distinctive clinicopathologic
features and gene usage analysis has indicated clonal
V.delta.1-J.delta.1 rearrangements associated with these
lymphomas..sup.125 Furthermore, the rare type of cutaneous
TCR.gamma..delta..sup.+ T cell lymphomas express V.delta.2 and
therefore appear to represent a clonal expansion of
TCR.gamma..delta..sup.+ T cells which normally reside in the
skin..sup.126 Other clonal TCR.gamma..delta. proliferations include
CD3.sup.+ TCR.gamma..delta..sup.+ large granular lymphocyte (LGL)
proliferations which comprise about 5% of all CD3.sup.+ LGL and
often show V.delta.1-J.delta.1 rearrangements..sup.127
[0196] The development of monoclonal antibodies towards framework
regions of TCR.gamma..delta. and more recently to specific V.delta.
gene segments has helped identify TCR.gamma..delta..sup.+ T cell
populations by flow cytometric analysis,.sup.15 but PCR clonality
studies are still required to identify whether these populations
represent clonal or polyclonal expansions..sup.128
Primer Design
[0197] The TCRD gene segments, consisting of eight V.delta., four
J.delta. and three D.delta. gene segments, show little or no
homology to each other and so segment-specific primers were
designed which would not cross-anneal with other gene segments.
Usage of V.delta.7 and V.delta.8 gene segments was considered too
rare to justify inclusion of primers for these segments and so,
following the general guidelines according to the invention for
primer design, a total of 16 primers were designed: 6 V.delta., 4
J.delta. and 5' and 3' of the 3 D.delta. gene segments (FIG. 9B).
All primers were designed for multiplex together in any
combination, but originally it was planned to have one tube (A)
with all V and all J primers which would amplify all the complete
V(D)J rearrangements and a second tube (B) with V.delta.2,
D.delta.2-5', D.delta.3-3' and J.delta.1 primers to amplify the
major partial rearrangements (V.delta.2-D.delta.3,
D.delta.2-D.delta.3 and D.delta.2-J.delta.1). Together these tubes
should amplify 95% of known rearrangements. The other primers
(D.delta.1-5.dbd., D.delta.3-5', D.delta.1-3' and D.delta.2-3')
could be used to amplify other D.delta.-J.delta., V.delta.-D.delta.
or D.delta.-D.delta. rearrangements, but were always intended to be
optional.
Results of Initial Testing Phase
[0198] All primer pair combinations were tested using polyclonal
DNA (tonsil and MNC). Most gave products of the expected size, but
some (D.delta.1-5', D.delta.1-3' and D.delta.2-3') gave no visible
product in combination with any other primer. Rearrangements
involving these primer regions are likely to be extremely rare and
so these, and D.delta.3-5', were excluded from subsequent testing.
Clonal cases for the six main rearrangements (V.delta.1-J.delta.1,
V.delta.2-J.delta.1, V.delta.3-J.delta.1, D.delta.2-D.delta.3,
V62-D.delta.3 and D.delta.2-J.delta.1) were tested initially in
monoplex PCR and then in multiplex tubes A and B (see above).
Serial dilutions of clonal DNA in polyclonal DNA (tonsil or MNC)
showed detection sensitivities of at least 5% in all cases.
However, in clonal cases with biallelic rearrangements, which were
clearly detected in single PCR reactions, the second, usually
larger, allele often failed to amplify on multiplexing. In
addition, it was found, using a different set of clonal cases that
several of the V.delta.2-J.delta.1 rearrangements failed to
amplify. A polymorphic site was subsequently identified at the
position of the original V.delta.2 primer;.sup.129 the frequency of
this polymorphism in the general population unknown, and so this
primer was redesigned to a new region of the V.delta.2 gene
segment, retested and found to amplify all cases. The problem with
the failure to amplify the second allele was overcome by increasing
the MgCl.sub.2 concentration from 1.5 mM to 2.0 mM.
[0199] We also tested the possibility of combining the two tubes
into a single multiplex reaction. Twelve clonal cases were tested,
which had a total of 21 gene rearrangements between them. A single
multiplex tube containing 12 primers (6 V.delta., 4 J.delta.,
D.delta.2-5' and D.delta.3-3') was used with ABI Gold buffer and
2.0 mM MgCl.sub.2 to amplify all the cases. All gene rearrangements
were indeed detected with a sensitivity of 0.5-10% by HD analysis
when diluted in polyclonal MNC DNA (Table 7). The only problem with
combining all TCRD primers in a single tube was the appearance of a
nonspecific band at about 90 bp in all amplifications, which was
not present when the two separate multiplex tubes were used. Since
the band was outside the size range of the TCRD products and did
not interfere with interpretation, it was not considered to be a
problem.
Results of General Testing Phase
[0200] The testing of the 90 Southern blot-defined samples in ten
laboratories raised some general issues about the TCRD
protocol:
[0201] Interpretation of some GS results was difficult. Because of
the large size range of products for the TCRD locus, there is no
classical Gaussian distribution for polyclonal samples (see FIG.
9C) and this, coupled with the low usage of TCRD in many samples
meant that in some cases it was hard to determine whether a sample
was polyclonal or clonal. The same problem did not arise with HD
analysis and so the recommendation is that GS should only be used
for TCRD with extreme care and awareness of the potential
problems.
[0202] The 90 bp nonspecific band was quite intense in some
laboratories, but less so in others. It appeared to be weaker when
using Buffer II rather than Gold buffer (confirmed by subsequent
testing) and is also sensitive to MgCl.sub.2 concentration,
becoming more intense as MgCl.sub.2 concentration increases. This
product has now been sequenced and found to be an unrelated gene
utilizing the D.delta.2 and J.delta.3 primers.
[0203] The results of the general testing of the 90 Southern blot
defined samples showed that the overall concordance of all the PCR
groups doing the testing was very high (95%). Of the 90 cases, six
were Southern blot positive for TCRD clonal rearrangements, five of
which were found to be clonal by PCR. The remaining case (DE-10, a
T-ALL with high tumor load) was found to be polyclonal by all labs.
Of the 84 Southern blot negative cases, 75 were found to be
polyclonal by PCR, four were found to be clonal and the remaining
five cases showed discordance between the GS and HD results. Of the
clonal cases, two (DE-2 and GBS-9) were T-rich B-NHLs with
presumably low tumor load and so the results may reflect the
increased sensitivity of PCR over Southern blotting. The other two
clonal cases (GBS-15 and ES-7) had high tumor load. Of the five
cases, which showed discrepancy between the GS and HE) results, one
(NL-1) was a difficult oligoclonal case, which caused problems for
several other loci. The remaining four were found to be polyclonal
by HD and clonal by GS. In three of the cases (NL-13, N-15 and
NL-18) this may reflect the greater sensitivity of GS over HD
analysis, but the remaining case (PT-1, a reactive lymph node) may
be attributed to "pseudoclonality" on GS analysis because of the
limited repertoire of TCRD usage in some samples.
Conclusion
[0204] In conclusion, the recommended protocol for detection of
TCRD gene rearrangements is a single tube assay containing 12
primers for detection of all major V.delta.(D)J.delta.,
V.delta.-D.delta., D.delta.-D.delta. and D.delta.-J.delta.
rearrangements using Buffer II and 2.0 mM MgCl.sub.2 to ensure
maximum specificity and detection. The preferred analysis method is
HD, but GS may be used with dare if consideration is given to the
problems of pseudoclonality caused by the limited usage of TCRD in
some samples. However, the use of multi-color GeneScanning (see
FIG. 13C) can be helpful in rapid recognition of the different
types of complete and incomplete TCRD gene rearrangements in the
different types of ALL. With these limitations in mind, TCRD can
nevertheless be a valuable target for the more immature T-cell
leukemias as well as TCR.gamma..delta..sup.+ T-cell
proliferations.
EXAMPLE 8
t(11;14) with BCL1-IGH Rearrangement
Background
[0205] The t(11;14)(q13;q32) is characteristic for mantle cell
lymphoma (MCL) because this cytogenetic reciprocal translocation
was observed in 60-70% of MCL cases and only sporadically in other
B-cell NHL..sup.130 The breakpoint region was originally cloned by
Tsujimoto et al (1983) and referred to as the BCL1-region..sup.131
However in only few cases with a cytogenetic t(11;14) a genomic
breakpoint in the BCL1-region was identified. Using fiber and
interphase FISH with probes covering the approximately 750 kb
11q13-BCL1 region, in almost all MCL (33 out of 34) a breakpoint
was observed and all breakpoints were confined to a region of 360
kb 5' of the cyclin D1 gene..sup.132, 133 In nearly half of MCL
cases (41%) the breakpoints were clustered within an 85 bp region
that was referred to as the major translocation cluster region,
BCL1-MTC..sup.130, 134, 135 In most if not all cases of MCL the
break at the IGH locus located at 14q32 involves the JH genes
juxtaposing the IGH-E.mu. enhancer to chromosome 11q13 sequences
and consequently resulting in transcriptional activation of the
cyclin D1 gene..sup.138 Cyclin D1 together with CDK4 phosphorylates
(and inactivates) pRB and allows for progression through the G1
phase of the cell cycle. Because cyclin D1 is silent in
B-lymphocytes and B-cell NHL other than MCL, and the presence of
this translocation correlates well with cyclin D1 expression, this
gene is considered to be the biological relevant target in
MCL..sup.138 Both expression of cyclin D1 and/or the presence of
t(11;14)(q13;q32) is used as an additional tool in the differential
diagnosis of NHL..sup.2 The gold standard detection strategy for
the presence of the t(11;14) that will identify almost all
breakpoints is interphase FISH using breakpoint-flanking probes in
fresh or frozen material.sup.133 as well as in archival
specimens..sup.137 However, a PCR based detection strategy for the
t(11;14) might be useful for e.g; residual disease monitoring. Many
groups have developed PCR based assays to detect the
BCL1/JH-breakpoints, in general using a consensus JH-primer in
combination with primers in the BCL1-MTC region that were all
located in a region of 392 bp..sup.54, 55 Breaks within the
BCL1-MTC region can occur upto 2 kb downstream of the MTC region,
but the majority of breakpoints are tightly clustered within an 85
bp segment, immediately downstream of the reported most 3'-primer
("primer B" in.sup.54, 134). Because breaks in this BCL1-MTC-region
account for only part of the breakpoints in the 11q13-BCL1 region
in MCL cases (41%), the PCR based strategy for t(11;14) seriously
impairs the diagnostic capability with an high rate of
false-negative results as compared to FISH.
[0206] The t(11;14)(q13;q32) has also been reported to be observed
in other B-cell proliferative diseases such as multiple myeloma
(20%), SLVL (30%), B-PLL (33%) and B-CLL (8%)..sup.130, 138, 139
One reason for the presence of the t(11; 14) in B-CLL in some
studies might be due to the incorrect classification of
B-CLL..sup.130 In myeloma the breakpoints are quite different from
those in MCL because (i) the frequency is much lower; (ii) most
breaks involve switch-class recombination sites; and (iii) although
all tested cases are located in the same 360 kb BCL1-region there
seems to be no preferential clustering within the BCL1-MTC region.
On the other hand, in all cases with a break the cyclin D1 gene is
activated. Of note, in a subgroup of multiple myelomas with a
IGH-switch-break myeov, an additional region in the 11q13-BCL1
region, is involved..sup.138
Primer Design
[0207] Based on the location of the reported most-far 5'-breakpoint
and available nucleotide sequences from the BCL1-MTC region
(GenBank accession number S77049), we designed a single BCL1 primer
(5'-GGATAAAGGCGAGGAGCATAA-3') in the 472-bp region 5' of this
breakpoint by using the primer design program OLIGO6.2 relative to
the consensus JH primer.
Results of Initial Testing Phase
[0208] Using the consensus JH-primer in combination with the single
BCL1-MTC-primer on a small series of MCL (n=5) previously
identified as positive with an in-house BCL1/JH-PCR using a similar
consensus JH18-primer (18 nt) and 5'-GCACTGTCTGGATGCACCGC-3' as
BCL1-MTC-primer, we initially compared both assays in parallel. In
contrast to the analysis of Ig/TCR gene rearrangements via GS
and/or HD analysis, the BCL1-JH PCR products (as for BCL2-JH
products) are identified via agarose gel electrophoresis using
ethidium bromide staining only. The results on the five positive
and two negative samples were identical except that the PCR
products were significantly weaker. To evaluate whether we could
increase the sensitivity of the PCR, we determined the effect of
different concentrations of MgCl.sub.2 and primers, and different
temperatures in a Stratagene-Robocycler PCR-machine (all other PCR
were done on ABI-480 or ABI-9700). Most intriguing was the
variation due to small changes in MgCl.sub.2 concentration. At 2.0
mM a weak nonspecific product of 550 bp became apparent whereas at
2.5 mM and higher this nonspecific product was very prominent in
all DNAs including non-template DNA controls. At lower
concentrations (less than 1.5 mM) no nonspecific fragments were
observed but the expected specific products were very weak.
Hybridizations with a BCL1-MTC-internal oligo-probe
(5'-ACCGAATATGCAGTGCAGC-3') did not show hybridization to this 550
bp product. PCRs with each of the primers separately revealed that
the 550 bp product could be generated by using the JH-consensus
primer only. In some MCL cases, in addition to the PCR-products
ranging from 150-350 bp (FIG. 10B), larger specific PCR-products
might be apparent due to annealing of the consensus JH-primer to
downstream JH5 and JH6 segments as described for BCL2/JH..sup.140
From the initial testing phase the most optimal PCR-conditions for
the BCL1-MTC/JH-PCR were: annealing temperature of 60.degree. C.,
2.0 mM MgCl.sub.2 and 10 pmol of each primer (for 35 PCR-cycles in
the ABI 9700).
[0209] To evaluate the specificity of the PCR on a larger series of
cases, the BCL1-MTC/JH-PCR was performed in three laboratories on
DNA from in total 25 cases MCL that were all previously identified
as positive with in-house BCL1/JH-PCR, and from 18 negative
controls. None of the negative cases revealed a PCR-product whereas
22 of 25 positive cases showed products of the expected size. In
the three cases that did not reveal a product on agarose-gel, a
product was detected with GS suggesting that the sensitivity is
lower when compared to in-house PCR.
[0210] The sensitivity of the PCR was evaluated by amplifying DNA
dilutions of a MCL in normal tonsillar DNA. A sensitivity between
10.sup.-3 and 10.sup.-4 was observed on agarose gel using the
developed PCR-primers. An in-house PCR performed in parallel on the
same samples was at least 10.times. more sensitive. Hybridizations
with the in-house BCL1-MTC-oligo-probe revealed a 10-100.times.
higher sensitivity of both PCRs. Dilutions with DNA of an
established cell line JVM2 (available through DSMZ;
http://www.dsmz.de) with an BCL1-MTC/JH4-breakpoint.sup.53 is used
as our standard positive control. As a negative control normal
tonsillar tissue or peripheral blood cells might be used, but
almost any non-MCL B-cell NHL should be suitable because of the
very low frequency of this aberration..sup.130
Results of General Testing Phase
[0211] To evaluate inter-laboratory variations for the detection of
breakpoints at the BCL1-MTC region, ten groups participated in the
analysis of DNA from a series of 90 histologically defined
malignant and reactive lymphoproliferations using the
BCL1-MTC/JH-PCR protocol. All cases were defined for their status
at the Ig and TCR loci using Southern hybridization techniques. Of
the 90 cases, seven were histologically characterized as MCL. All
seven MCL cases were shown to have a clonal IGH rearrangement by
Southern hybridization. Assessment of rearrangements within the
BCL1-MTC-region at chromosome 11q13 by either Southern
hybridization or FISH was not performed in all cases. In six of the
seven MCL cases the PCR-product was identified in all ten
laboratories. In MCL case NL-15 in six of the laboratories the
expected 1.8 kb PCR product was identified. This particular case
carries an exceptional breakpoint with an uncommon large
PCR-product (normally ranging from 150 to 350 bp) and represents
the 3'-most-far detectable BCL1-MTC-breakpoint to our knowledge. In
two of six labs the PCR product was observed but initially
considered as nonspecific because of its uncommon size. In ES-4,
characterized histologically as MCL in none of the ten labs a
PCR-product could be detected suggesting that this case carries a
breakpoint outside the BCL1-MTC. It should be stressed that the MCL
cases submitted to this series for the general testing phase were
selected and thus are expected to carry breaks at the BCL1-MTC
region at an higher incidence than normal. Importantly, except for
one single case (FR-1), in all 83 other non-MCL cases including 16
cases that were histologically characterized as B-CLL, no
BCL1-MTC/JH-PCR product was detected in any laboratory. In case
FR-1 histologically characterized as B-CLL, in three of the ten
labs a product was identified indicating that the number of cells
with this break is low. The IGH status determined by Southern blot
analysis revealed that this sample was composed of 90% clonal
B-cells in good agreement with the histological examination.
PCR-based B-cell clonality analyses for IGH and IGK (sensitivity of
approximately 1%) revealed a single clone and Southern blot
analysis for IGK showed a single major IGK rearrangement only. In
addition, Northern blot analysis for expression of cyclin D1 did
not show overexpression. All these data suggested that the very
small number (less than 1%) t(11;14)-positive cells represent
either (i) a subclone derived from the B-CLL, (ii) an independent
second B-malignancy or (iii) normal B-cells as described for
t(14;18)-positive B-cells in normal individuals..sup.140 However,
with the available data of this patient at present we can not
discriminate between these three alternatives. In summary, the
analysis by the ten laboratories illustrates the high specificity
of the BCL1-MTC/JH-PCR strategy.
[0212] To evaluate the presence of possible false-negative cases
due to the relative low sensitivity of the PCR, in one laboratory
the previously described in-house PCR (with about 10-fold higher
sensitivity) was performed on DNA of all 90 cases and the PCR
products of both assays were also hybridized with an
internal-BCL1-MTC oligo-probe that increases the sensitivity
another 10-100-fold. This analysis revealed no PCR products in
other cases.
Conclusion
[0213] We conclude that also the sensitivity of the BCL1-MTC/JH PCR
(between 10.sup.-3 and 10.sup.-4) is sufficiently high for the
detection of the BCL1-MTC /JH-breakpoint in diagnostic material.
The results of this approach are very encouraging and suggest that
the definition of common approaches and reaction conditions can
minimize erroneous results. However, it should be remembered that
maximally about 50% of the t(11;14) breakpoints in MCL will be
detected and that for diagnosis additional detection tools are
recommended.
EXAMPLE 9
t(14;18) with BCL2-IGH Rearrangement
Background
[0214] The t(14;18) is one of the best characterized recurrent
cytogenetic abnormalities in peripheral B cell lymphoproliferative
disease..sup.141 It is detectable in up to 90% of follicular
lymphomas and 20% of large cell B-cell lymphomas depending upon the
diagnostic test used..sup.142 As a consequence of the translocation
the BCL2 gene from 18q32 is placed under the control of the strong
enhancers of the IGH locus resulting in deregulation of its normal
pattern of expression..sup.143, 144 BCL2 is located on the outer
mitochondrial membrane and its normal function is to antagonize
apoptosis and when deregulated it is intimately involved in the
pathogenesis of the tumor..sup.145-148 As a consequence of this
role in pathogenesis the t(14;18) provides an ideal target for both
diagnosis and molecular monitoring of residual disease.
[0215] The IGH locus is located at 14q32.3 with the V.sub.H regions
lying telomeric and the D.sub.H, J.sub.H and constant regions
placed more centromeric. The transcriptional orientation is from
telomere to centromere with enhancers located 5' of the V regions
and between each of the constant regions. The most common form of
the translocation involves the process of VDJ recombination and one
of the six germline J.sub.H regions is closely opposed to BCL2.
Most PCR based detection strategies have utilized a consensus
J.sub.H primer that will detect the majority of
translocations..sup.149, 150 In contrast to the IGH locus, the
pattern of breaks in BCL2 is more complicated. BCL2 is located on
chromosome 18q21 and is orientated 5' to 3' from centromere to
telomere. The majority of breakpoints fall within the 150 bp MBR
located in the 3' untranslated region of exon 3..sup.151 As a
consequence of the translocation, the S.mu. enhancer located 3' of
the J.sub.H regions is placed in close proximity to the BCL2 gene
leading to its deregulation. As more translocations have been
investigated it has become apparent that there are a number of
other breakpoint regions which must be taken into account for an
efficient PCR detection strategy. Positioned 4 kb downstream of the
MBR is a further breakpoint region, the 3' MBR subcluster,
encompassing a region of 3.8 kb..sup.152 The mcr is located 20 kb
3' of the MBR and covers a region of 500 bp..sup.153 However,
though analogous to the MBR, the mcr is more extensive than was
initially envisaged and a region 10 kb upstream of the mcr, the 5'
mcr subcluster, has been described..sup.154, 155 In addition to
these classical breakpoints a number of variant translocations are
described where the breaks occur 5' of BCL2..sup.158 These are,
however, rare and thus can not be taken into account using a PCR
based detection strategy.
[0216] There is no single gold standard detection strategy for the
t(14;18) and a combination of cytogenetics and Southern blotting
have been generally used..sup.157, 158 Interphase FISH detection
strategies offer an applicable alternative that have the potential
to pick up more translocations..sup.159 In contrast DNA based fiber
FISH has been very informative for defining variant translocations
but is unsuitable for routine application..sup.160 For molecular
diagnostic laboratories PCR based detection strategies offer rapid
results, are generally applicable and can be used for residual
disease monitoring. However, the primers commonly used have been
derived on an ad hoc basis and have not been designed to take into
account recent information on the molecular anatomy of the
breakpoints. As a consequence when compared to gold standard
approaches, PCR based techniques only detect up to 60% of
translocations which seriously impairs the diagnostic capability of
PCR. Compounding this high percentage of false negative results is
the problem of false positive results arising from contamination
from other samples and previously amplified PCR products.
Primer Design
[0217] We initially evaluated a two tube multiplex system, one tube
designed to detect breakpoints within the MBR and a second tube
used to identify breakpoints outside this region. The MBR strategy
contained three primers MBR1, MBR2 and the consensus JH primer. The
second multiplex reaction contained five primers, MCR1, MCR2,
5'mcr, 3' MBR1 and the consensus JH (FIG. 11A) and was designed to
detect breakpoints within the mcr, 5'mcr and 3' MBR regions.
Results of Initial Testing Phase
[0218] The evaluation of these primers was performed in three
laboratories on DNA derived from a total of 124 cases of follicular
lymphoma known to carry a t(14; 18). 109 cases (88%) were
identified with an BCL2-IGH fusion, 83/124 (67%) were positive
using the MBR multiplex and 26/124 (21%) were positive using the
non-MBR multiplex strategy. In 15/124 (12%) cases there was no
amplifiable PCR product. Further examination of the cases
identified with the non-MBR multiplex showed that 11 (9%) had a
breakpoint within the mcr, five cases (4%) within the 5'mcr and
10/124 (8%) within the 3'MBR.
[0219] To further investigate the value of this set of primers for
the detection of breakpoints within the 5'mcr and 3'MBR sub-cluster
regions a series of 32 cases of t(14;18) positive follicular
lymphomas known to be germline at the MBR and mcr by Southern
hybridization were analyzed in one laboratory. Five of the cases
had breakpoints within the 5'mcr (260-490 bp) and were amplified
using both the 5'mcr primer in isolation and with the multiplex
reaction. None of the remainder of cases showed a positive result.
Of the series of 32 cases, nine were already known to have
breakpoints within the 3'MBR region and the multiplex approach was
able to detect 5/9 of these cases.
[0220] In order to improve the sensitivity of the assay within this
region we designed three further primers that spanned the 3'MBR
sub-cluster region; 3'MBR2, 3'MBR3 and 3'MBR4 and combined them
with 3'MBR1 and the consensus JH in an additional multiplex
reaction; 3'MBR multiplex FIG. 11). This new approach confirmed
that eight of the 32 cases were positive but missed the ninth case.
The primers were then used individually and in this experiment 11
of the 32 cases were positive. The breakpoints were distributed as
follows; 2/11 cases had a breakpoint present between primer 3'MBR1
and 3'MBR2, 3/11 cases between primers 3'MBR2 and 3'MBR3, 2/11
cases between primers 3'MBR3 and 3'MBR4 and the remaining four
cases amplified using primer 3'MBR4 and were distributed 200-1000
bp 3' of this primer. In this series of cases there were three
false negative results using the 3'MBR multiplex. One of the cases
was a true false negative where the break occurred in the middle of
the 3'MBR, in proximity to an Alu repeat sequence. The
translocation was detected using the 3'MBR3 primer when used in
isolation and a product of 450 bp was generated suggesting a
reduced sensitivity of the multiplex. The remaining two false
negative cases generated products larger than 1000 bp with the
3'MBR4 primer, placing them in the far 3'MBR not fully covered by
this approach. Further improvement in the sensitivity of the 3'MBR
assay has been achieved following the general testing phase of this
study. Substituting primer 3'MBR3 with a new downstream primer
5'-GGTGACAGAGCAAAACATGAACA-3' (see FIG. 11A) significantly improved
both the sensitivity and specificity of the 3'MBR assay.
[0221] Based on this, the 3'MBR multiplex was incorporated into our
diagnostic strategy. Analysis of the Southern blot defined cases
was therefore carried out using the three tube multiplex system
presented in FIG. 11A.
Results of General Testing Phase
[0222] Inter-laboratory variations feature significantly in
diagnostic PCR strategies. To evaluate this, 11 groups participated
in an extensive external quality control exercise. DNA was
extracted from a series of 90 histologically defined malignant and
reactive lymphoproliferations were analyzed using the t(14;18)
multiplex protocol (FIGS. 11B, C, and D). All cases were defined
for their status at the Ig and TCR loci using Southern
hybridization techniques. Karyotypic confirmation of the t(14;18)
was not available on this series. We therefore adopted an approach
requiring greater than 70% concordance between members of the
network for acceptance of the t(14;18). Of the 90 cases, 11 were
characterized histologically as follicular lymphoma. All 11 cases
were shown to have a clonal IGH rearrangement by Southern
hybridization. Assessment of rearrangements within the BCL2 gene
was also performed by Southern hybridization using specific probes
to the MBR, mcr and 3'MBR in 10/11 cases. 4/10 cases showed a
rearrangement within the MBR that was concordant with the PCR
result. A single case, GBS-7, shown to be mcr multiplex positive,
gave an inconclusive SB result with the mcr probe.
Immunophenotypically this case demonstrated two distinct clonal
populations, representing approximately 5% and 15% of the original
diagnostic material. The discrepancy between the two techniques in
this case probably represents the reduced sensitivity of SB
compared with PCR. There was no evidence of a 3'MBR rearrangement
in any of the remaining cases by SB.
[0223] Of the six SB negative FCL cases, a single case, ES-7,
showed a t(14;18) using the MBR multiplex. 5/11 FCL cases showed no
evidence of a t(14;18) by either SB or PCR. A t(14;18) was detected
in two further cases by PCR; FR-6, a case of DLBCL showed an MBR
breakpoint and was identified by all 11 laboratories, this finding
is compatible with previous studies that have detected a t(14; 18)
in 20-40% of DLBCL cases..sup.161, 162 Using the 3'MBR multiplex,
10/11 laboratories reported a positive result for sample ES-12,
this was a case of Hodgkin's disease which contained very few B
cells. It is difficult to explain this result in the absence of an
IGH rearrangement by Southern blotting. Contamination or incorrect
labeling of the sample at source is the most likely
explanation.
[0224] Overall there was excellent concordance throughout the
network, although small numbers of both false positive and false
negative results were encountered. Overall 12 false positive
results were identified, representing less than 0.4% (12/3036) of
the total number of analyses. These were reported by five
laboratories and involved six of the samples. The majority of the
false positives (9/12) were found in three cases. Five false
negative results, representing a 6% (5/88) failure rate, were
reported by three laboratories, ES-7 was not detected by two
laboratories, three further groups within the network commented
that this case had shown weak amplification signals with the MBR
multiplex. The remaining three false negative cases were reported
in isolation by individual laboratories. The results of diagnoses
using this approach are very encouraging and suggest that the
definition of common approaches and reaction conditions can
minimize erroneous results.
Conclusion
[0225] In conclusion, we have designed and evaluated a robust
three-tube multiplex PCR in order to maximize the detection of the
t(14;18). This strategy is capable of amplifying across the
breakpoint region in the majority of cases of FCL with a
cytogenetically defined translocation. Although the sensitivity of
this strategy is lower than conventional single round or nested PCR
approaches, it is still perfectly acceptable for diagnostic
procedures. The widespread adoption of standardized reagents and
methodologies has helped to minimize inaccurate results within this
large multi-center network. However, it is noteworthy from the
general testing phase of this study that it is impossible to detect
a t(14;18) in all cases. This is certainly influenced by additional
molecular mechanisms capable of deregulating the BCL2
gene..sup.163, 164
EXAMPLE 10
Use of DNA Extracted from Paraffin-Embedded Tissue Biopsies and
Development of Control Gene Primer Set
Background
[0226] Fresh/frozen tissue is considered to be the ideal sample
type for extraction of DNA for use in PCR-based clonality analysis.
However, fresh/frozen material is not always available to
diagnostic laboratories and in many laboratories throughout Europe,
paraffin-embedded tissue samples constitute the majority of
diagnostic biopsies submitted for analysis. DNA extracted from
paraffin-embedded material is often of poor quality and so PCR
protocols need to be evaluated for use with these sample types
before they can be widely used in diagnostic laboratories.
[0227] The integrity of DNA extracted from paraffin-embedded
samples and its amplification by PCR are affected by a number of
factors such as thickness of tissue, fixative type, fixative time,
length of storage before analysis, DNA extraction procedures and
the co-extraction of PCR inhibitors..sup.165-172 Ten percent
neutral buffered formalin (10% NBF) is the most commonly used
fixative, although laboratories also use a number of other
fixatives, including unbuffered formalin and Bouins. The use of 10%
NBF permits the amplification of DNA fragments of a wide range of
sizes whereas Bouins appears to be the least amenable for use in
PCR analysis..sup.167, 168, 171, 173 The integrity of DNA fragments
extracted from paraffin-embedded samples also depends on the length
of time the blocks have been stored with the best results usually
obtained from blocks less than 2 years old, while blocks over 15
years old tend to yield very degraded fragments..sup.174
Primer Design
[0228] Initially, five pairs of control gene PCR primers were
designed to amplify products of exactly 100, 200, 400, 600 and
1,000 bp in order to assess the quality of DNA submitted for
analysis. The target genes were selected on the basis of having
large exons with open reading frames to reduce the risk of
selecting polymorphic regions and the primers were designed for
multiplex usage in the standardized protocols. The following target
genes were selected: human thromboxane synthase gene (TBXAS1, Exon
9; GenBank Accession No D34621), human recombination activating
gene (RAG1, Exon 2; GenBank Accession No M29474), human
promyelocytic leukemia zinc finger gene (PLZF, Exon 1; GenBank
Accession No AF060568), and human AF4 gene (Exon 3; GenBank
Accession No Z83679, and Exon 11; GenBank Accession No Z83687).
Results of Initial Testing Phase
[0229] The primer pairs were tested in separate reactions and
subsequently in multiple reactions using high molecular weight DNA.
Due to the large size range of the products (100 to 1,000 bp), it
was necessary to vary the ratio of primer concentrations to obtain
bands of equal intensities in the multiplex reactions. However, it
proved extremely difficult to be able to amplify all the bands
reproducibly and it was decided that the 1,000 bp product was
probably unnecessary, since all the PCR protocols according to the
invention give products of less than 600 bp. It was therefore
decided to exclude the 1,000 bp product in order to improve the
reproducibility of the assay. By increasing the MgCl.sub.2
concentration to 2 mM and adding the primers in a 1:1:1:2 ratio, it
was possible to reproducibly amplify four bands (100, 200, 400 and
600 bp) of equal intensity from high molecular weight DNA samples.
However, for DNA extracted from paraffin blocks, it was thought
that an extra size marker at 300 bp would be extremely informative
and that the 600 bp marker might not be necessary. Using the gene
sequence for the 1,000 bp marker (PLZF), primers were redesigned to
generate a 300 bp product. These were tested successfully both in
monoplex reactions and in multiplex reactions combining the 100,
200, 300, 400 and 600 bp primers (see FIG. 12A).
[0230] Thus two primer sets are available for assessing the quality
of DNA for amplification: The 100, 200, 300 and 400 bp primers used
at 2.5 pmol each can be used for assessing DNA from
paraffin-embedded tissues. The addition of the 600 bp primers at 5
pmol allows this set to be used to check the quality of any DNA
sample for use with the primers and protocols according to the
invention. Both primer sets can be used with ABI Buffer II and 2.0
mM MgCl.sub.2 under standardized amplification conditions. Products
can be analyzed on 6% PAGE or 2% agarose (see FIG. 12B).
Results of Leneral Testing Phase
[0231] Forty five paraffin-embedded biopsies were collected
corresponding to 30 of the B-cell malignancies, eight of the T-cell
malignancies and seven of the reactive lymphoproliferations
submitted as fresh/frozen tissue samples. The age of the paraffin
blocks as well as the methods of fixation and embedding of the
samples varied between National Networks. The ES samples were
submitted as pre-cut sections, NL-14, 15 and 16 were submitted as
DNA samples and the remaining biopsies were submitted as paraffin
blocks. Five sections (10 .mu.m each) were cut from the paraffin
blocks and DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN)
following the manufacturer's protocol for isolation of genomic DNA
from paraffin-embedded tissue. This method of DNA extraction was
chosen since the kit can be used to rapidly extract good quality
DNA from blood, fresh/frozen tissue and paraffin-embedded tissue
and thus enables the parallel processing of a variety of sample
types with assured quality control. Numerous protocols for
extraction of DNA from paraffin-embedded tissue for PCR analysis
have been published..sup.171, 172, 175-177 Many of these aim to
reduce DNA degradation and co-extraction of PCR inhibitors, but
many of these methods require prolonged extraction procedures and
can be unsuitable for use in the routine diagnostic
laboratory..sup.168, 178, 179
[0232] DNA sample concentration and integrity were estimated by
spectrophotometry and by comparison of sample DNA with known
standards on agarose gel electrophoresis. DNA samples (100 ng) were
then analyzed for integrity and amplifiability using the control
gene PCR primers (100-400 bp) and assessed for clonality at all
target loci using the PCR protocols.
[0233] In the control gene PCR reaction of 24/45 cases the
amplified products were at least 300 bp, whereas in the remaining
21 samples the amplified products were 200 bp or less. No clear
correlation between the quality of the DNA and the age of the block
or fixation method could be demonstrated. Therefore it is likely
that a combination of factors is responsible for the DNA quality in
these samples.
[0234] The DNA samples were evaluated for clonality using the 18
multiplex PCR reactions and were analyzed by both HD and GS. The
number of paraffin samples showing clonality and translocations at
the nine target loci were compared with the corresponding
fresh/frozen sample data. In samples with control gene PCR products
of up to 200 bp, the overall detection of clonality at the nine
target loci was 9/55 (16%). Of the 46 missed rearrangements, 45
could be explained by the fact that the expected clonal PCR
products had a molecular weight higher than the maximum size
amplified by the sample in the control gene PCR. The remaining
sample (PT-9) amplified-to 100 bp in the control gene PCR but the
expected 81 bp TCRG clonal product was not detected. In samples
with control gene PCR products of at least 300 bp, the overall
detection of clonality at the nine target loci was 42/55 (76%). Of
the 13 missed rearrangements, five could again be explained by the
fact that the expected clonal PCR products were larger than the
maximum size amplified by the sample in the control gene PCR. The
remaining eight missed rearrangements could not be explained
directly by the quality of the DNA. One false positive clonal
result (GBN-9; IGL) was detected in a reactive lymph node which may
represent pseudoclonality.
[0235] PCR inhibitors are known to be present in DNA extracted from
paraffin samples. Dilution of the DNA sample may reduce the
concentration of these inhibitors to levels that allow successful
amplification to occur. To investigate the effect of diluting DNA
samples on the efficiency of amplification, four different
concentrations of DNA were tested in the control gene PCR reaction:
5, 50, 100 and 500 ng. We observed that dilution of the DNA samples
has a significant effect on the size of the PCR products in the
control gene PCR. Overall, 24/45 cases (53%) showed an increased
efficiency of amplification when diluted from 100 ng to 50 ng. The
optimal DNA concentration appears to be between 50 to 100 ng
whereas the use of 500 ng appears to inhibit the amplification of
large products (300 bp or above). Although the use of 5 ng of DNA
gives acceptable results with the control gene PCR, this can lead
to false positivity in PCR-based clonality assays due to the low
representation-of total lymphoid cell DNA..sup.180, 181 More
importantly, 5 ng of DNA has no advantage over a dilution to 50 ng
of DNA
[0236] To assess whether the use of 50 ng of DNA would also
increase the detection of clonality, all the samples were retested
at the IGH V-J locus using this DNA concentration. The number of
clonal rearrangements detected in the three IGH V-J tubes using 100
ng of DNA was 12, compared with 23 using the corresponding
fresh/frozen samples. The overall detection of clonality at this
locus increased to 17 out of 23 when 50 ng of DNA was used, with an
additional 9 FR1, 6 FR2 and 4 FR3 clonal products being detected.
Thus dilution of the DNA can increase the detection of clonal
products, presumably because of dilution of PCR inhibitors.
Logically, dilution of DNA is only likely to improve both control
gene PCR results and the detection of clonality, if PCR inhibitors
are present, not if the DNA sample is highly degraded. Therefore it
is recommended that at least two dilutions of DNA are tested using
the control gene PCR and that the dilution that gives the better
result is used in subsequent clonality analysis.
[0237] Nine clonal rearrangements remained undetected after initial
analysis, which could not be explained by DNA quality (TCRG in PT-9
and NL-11; TCRB in-GBS-4; TCRD in NL-15; IGK in GBN-4, NL-4 and
NL-5; IGH V-J.sub.H in GBS-6 and GBS-8). These samples were
retested using 50 ng of DNA, but only one sample (GBS8; IGH) showed
improved detection, suggesting that other, unknown, factors can
prevent amplification of specific targets in a small number of
cases. However, it should be noted that for seven of these samples
(NL-11, GBS-4, NL-15, GBN-4, NL-5, GBS-6 & GBS-8) clonal
products were detected in at least one other locus. This
demonstrates that testing for clonality at multiple target loci
increases the likelihood of detecting clonal lymphocyte
populations.
Conclusion
[0238] In conclusion, the protocols as provided herein work well
with DNA extracted from paraffin-embedded material provided that
the DNA can amplify products of 300 bp or more in the control gene
PCR. Two concentrations of DNA are preferably tested in the control
gene PCR and the more `amplifiable` concentration should be used in
further testing, although with the proviso that concentrations of
DNA less than 20 ng may contribute to the detection of
pseudoclonality due to the low representation of target lymphoid
DNA..sup.180, 181 Overall the data show that assessment of DNA
quality using the control gene PCR provides a good indication of
the suitability of the DNA for clonality analysis using the
protocols provided. It is also important to note that the control
gene PCR will give no indication of the amount of lymphoid cell DNA
present in the sample and therefore good quality DNA may still
produce negative results for clonality analysis. To ensure
monoclonal results are reproducible (and to avoid potential
pseudoclonality)), all clonality assays, particularly using
paraffin-extracted DNA, are preferably performed in duplicate and
analyzed by HD and GS, wherever possible.
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52: 104-110. TABLE-US-00001 TABLE 1 B, T, and NK lineage of
lymphoid malignancies.sup.a Chronic Non-Hodgkin lymphomas ALL
lymphocytic extra- Multiple Lineage childhood adult leukemias nodal
nodal skin myeloma B 82-86% 75-80% 95-97% 95-97% 90-95% 30-40% 100%
T 14-18% 20-25% 3-5% 3-5% 5-10% 60-70% 0% NK <1% <1% 1-2%
<2% <2% <2% 0% .sup.aSee Van Dongen et al. 1991.sup.1,
Jaffe et al. 2001.sup.2, and Van Dongen et al. 2002.sup.3
[0420] TABLE-US-00002 TABLE 2 Estimated number of non-polymorphic
human V, D, and J gene segments that can potentially be involved in
Ig or TCR gene rearrangements.sup.a Gene segment IGH IGK IGL TCRA
TCRB TCRG TCRD V segments functional (family) 44 (7) 43 (7) 38 (10)
46 (32) 47 (23) 6 (4) 8 rearrangeable (family) 66 (7).sup.b 76 (7)
56 (11) 54 (32) 67 (30) 9 (4) 8 D segments rearrangeable (family)
27 (7) -- -- -- 2 -- 3 J segments functional 6.sup.c 5.sup.d 4 53
13 5 4 rearrangeable 6.sup.c 5.sup.d 5.sup.e 61 13 5 4 .sup.aOnly
non-polymorphic gene segments with a suitable RSS are included in
this table. .sup.bThis estimation does not include the recently
discovered (generally truncated) VH pseudogenes, which are
clustered in three clans .sup.cThe six JH gene segments are highly
homologous over a stretch of .about.20 nucleotides, which is
sufficient for the design of a consensus primer. .sup.dThe J.kappa.
segments have a high homology, which allows the design of 2 to 3
J.kappa. consensus primers. .sup.eFive of the seven J.lamda. gene
segments have a suitable RSS.
[0421] TABLE-US-00003 TABLE 3 Standardized PCR protocol Reaction
conditions. buffer: ABI Buffer II or ABI Gold Buffer 50 .mu.l final
volume 100 ng DNA 10 pmol of each primer (unlabeled or 6-FAM
labeled) (irrespective of total numbers of primers in each
multiplex PCR tube) dNTP: 200 .mu.M final concentration MgCl.sub.2:
1.5 mM final concentration (to be optimized per target) Taq
enzyme.sup.a: 1 U in most tubes; 2 U in tubes with many primers
(>15) Cycling conditions pre-activation 7 min. at 95.degree. C.
annealing temperature: 60.degree. C. cycling times: "classical"
"newer" PCR equipment PCR equipment denaturation 45 sec. 30 sec.
annealing .gtoreq.45 sec. .gtoreq.30 sec. extension 1.30 min.
.gtoreq.30 sec. final extension .gtoreq.10 min. .gtoreq.10 min.
number of cycles: 35 hold 15.degree. C. (or room temperature)
.sup.aAmpliTaq Gold (Applied Biosystems, Foster City, CA) was used
in combination with 1x ABI Buffer II or 1x ABI Gold Buffer (Applied
Biosystems), depending on the target.
[0422] TABLE-US-00004 TABLE 4 Standardized protocol for
heteroduplex analysis of PCR products PCR product preparation tube
with 10-20 .mu.l of PCR product denaturation of PCR product: 5 min.
at 95.degree. C. re-annealing of PCR product: 60 min. at 4.degree.
C. Electrophoresis conditions (non-commercial polyacrylamide gels)
gel: 6% non-denaturing polyacrylamide (acrylamide: bisacrylamide
29:1) buffer: 0.5 .times. TBE loading buffer: 5 .mu.l ice-cold
non-denaturing bromophenol blue loading buffer electrophoresis:
typically 2-3 hours at 110 V or overnight at 40-50 V.sup.a
Electrophoresis conditions (commercial polyacrylamide gels) gel:
non-denaturing polyacrylamide (e.g. BioRad PreCast Gel System or
Amersham Pharmacia Biotech Gene Gel Excel Kit) buffer: 1 .times.
TBE loading buffer: ice-cold non-denaturing bromophenol blue
loading buffer electrophoresis: 1, 5 hours at 100 V Visualization
staining: 5-10 min. in 0.5 .mu.g/ml EtBr in H.sub.2O
destaining/washing: 2x 5-10 min. in H.sub.2O visualization: UV
illumination alternative: silver staining using Amersham Pharmacia
Biotech DNA Silver stain kit .sup.aVoltage and electrophoresis time
depend on PCR amplicon sizes, thickness of polyacrylamide gel, and
type of PCR equipment, and should be adapted accordingly.
[0423] TABLE-US-00005 TABLE 5 Standardized protocol for
GeneScanning of PCR products A. Gel-based sequencers PCR product
preparation 1. PCR product dilution: initially 1:10 in formamide or
H.sub.2O (can be altered if fluorescent signal is outside optimal
range; see electrophoresis conditions) 2. sample volume: 2 .mu.l
diluted PCR product 3. loading buffer volume: 0.5 .mu.l blue
dextran loading buffer + 0.5 .mu.l TAMRA internal standard + 2
.mu.l deionized formamide 4. denaturation of PCR product: 2 min. at
95.degree. C. or higher temperature 5. cooling of PCR product at
4.degree. C. Electrophoresis conditions 6. gel: 5% denaturing
polyacrylamide 7. buffer: 1 .times. TBE 8. electrophoresis: 2-3.5
hours.sup.a (see Table 25) 9. optimal fluorescent signal intensity:
600-4,000 fluorescent units (373 platforms) 400-7,000 fluorescent
units (377 platforms) B. Capillary sequencers (to be optimized per
sequencer) PCR product preparation 1. 1 .mu.l PCR product (volume
of PCR product or sampling times can be altered if fluorescent
signal is outside optimal range; see electrophoresis conditions) 2.
sample volume: 1 .mu.l PCR product + 9.5 .mu.l (Hi-Di) formamide +
0.5 .mu.l ROX-400 heteroduplex analysis internal standard 3.
denaturation of PCR product: 2 min. at 95.degree. C. or higher
temperature 4. cooling of PCR product at 4.degree. C. for an hour
Electrophoresis conditions 5. gel: 3100 POP4 polymer 6. buffer:
1.times. 3100 buffer with EDTA 7. electrophoresis: 45 minutes.sup.b
8. optimal fluorescent signal intensity: up to 10,000 fluorescent
units .sup.aElectrophoresis time depends on amplicon sizes and on
employed platform. .sup.bFor 36 cm capillary; time taken depends on
capillary used.
[0424] TABLE-US-00006 TABLE 6 Sensitivity of detection of clonal
TCRB rearrangements Involved primer Sensitivity of detection TCRB
pair Clonal Size of multiplex tube V J Control PCR product single
PCR.sup.a PCR tube A V.beta.2 J.beta.1.2 patient 261 nt 1-5% 5%
V.beta.2 J.beta.1.3 patient 267 nt 5% 5% V.beta.2 J.beta.1.6
patient 267 nt <5% V.beta.7 J.beta.2.2 patient 254 nt 10%
V.beta.8a J.beta.1.2 Jurkat 267 nt 0.1% 0.5-1% V.beta.8a J.beta.2.7
patient 264 nt 10% V.beta.10 J.beta.2.7 PEER 263 nt 20%
V.beta.3/12a/13a/15 J.beta.1.6 patient 278 nt <5% 5%
V.beta.3/12a/13a/15 J.beta.2.7 patient 286 nt 10% V.beta.17
J.beta.2.7 RPMI- 260 nt 10% 8402 V.beta.17 J.beta.1.1 patient 260
nt 1% 10% V.beta.18 J.beta.1.2 DND41 261 nt 1% 10% V.beta.22
J.beta.1.1 patient 265 nt 0.1% 10% V.beta.8b/23 J.beta.1.2 H9 257
nt 0.1% 0.5% V.beta.24 J.beta.1.5 RPMI- 264 nt 0.5% 10% 8402 tube B
V.beta.2 J.beta.2.1 Molt-4 267 nt 5% 5% V.beta.1/5 J.beta.2.1
patient 266 nt 5% 1-5% V.beta.6a/11 J.beta.2.1 patient 265 nt 1% 5%
V.beta.6a/11 J.beta.2.5 patient 258 nt 5% V.beta.7 J.beta.2.3 PEER
271 nt <5% V.beta.8a J.beta.2.1 patient 293 nt 0.1% 1%
V.beta.3/12a/13a/15 J.beta.2.1 patient 258 nt 5% 10%
V.beta.3/12a/13a/15 J.beta.2.3 patient 258 nt <5% V.beta.16
J.beta.2.1 patient 258 nt 0.5% 10% V.beta.17 J.beta.2.5 CML-T1 270
nt 0.1-1% 1% V.beta.21 J.beta.2.3 patient 282 nt 0.5% <10% tube
C D.beta.1 J.beta.1.1 patient 304 nt 0.10% <5% D.beta.1
J.beta.1.2 patient 306 nt 5% 5% D.beta.1 J.beta.1.4 patient 310 nt
5-10% D.beta.1 J.beta.1.6 patient 320 nt 20% D.beta.1 J.beta.2.1
patient 309 nt 5% 20% D.beta.1 J.beta.2.7 patient 307 nt <5%
D.beta.1 J.beta.2.5 patient 310 nt <1% D.beta.2 J.beta.1.4
patient 182 nt <1% D.beta.2 J.beta.2.1 patient 185 nt 1% <5%
D.beta.2 J.beta.2.5 patient 191 nt 5% .sup.aThe dilution experiment
for assessing the sensitivity of the single PCR was not performed
in each case.
[0425] TABLE-US-00007 TABLE 7 Sensitivity of detection of clonal
TCRD gene rearrangements Clonal control Sensitivity of TCRD sample
detection by rearrangement (approximate size) heteroduplex
V.delta.1-J.delta.1 patient (200 nt) 5% patient (190 nt) 1-5%
patient (200 nt) 5% V.delta.2-J.delta.1 patient (200 nt) 5% patient
(220 nt) 5% patient (210 nt) 5% V.delta.2-J.delta.3 patient (220
nt) 5% V.delta.3-J.delta.1 patient (270 nt) 5% V.delta.6-J.delta.2
Loucy (210 nt) 0.5% patient (210 nt) 10% D.delta.2-J.delta.1 Loucy
(150 nt) 0.2% patient (160 nt) 0.5% patient (135 nt) 0.5%
D.delta.2-J.delta.3 patient (150 nt) 5% D.delta.2-D.delta.3
NALM-16(170 nt) 1% patient (200 nt) 1% patient (190 nt) 0.5%
patient (170 nt) 0.5% V.delta.2-D.delta.3 REH (240 nt) 5-10%
NALM-16 (230 nt) 1-5% patient (250 nt) 5%
[0426] TABLE-US-00008 TABLE 8 Concordance between multiplex PCR
results and Southern blot (SB) analysis results (PCR/SB) on Ig/TCR
gene rearrangements per (sub)category of included frozen samples
Diagnosis IGH.sup.a IGK IGL TCRB TCRG TCRD pre-follicular (n = 8)
C.sup.b: 8/8 C: 8/8 C: 4/4 C: 2/4.sup.b C: 0/0 C: 0/0 P.sup.b: 0/0
P: 0/0 P: 4/4 P: 4/4 P: 8/8 P: 8/8.sup.e B-CLL (n = 16) C: C: 16/16
C: 5/5 C: 1/1 C: 0/0 C: 2/2 15/16 P: 0/0 P: 9/11 P: 15/15 P: P: P:
0/0 16/16 14/14 (post-)follicular C: C: C: 3/5 C: 2/4 C: 0/1 C: 0/0
(n = 25) 22/25.sup.b 19/24.sup.c P: P: P: P: P: 0/0 P: 0/1 19/20
21/21.sup.d,e 22/24 24/25.sup.e All B-cell malignancies C: C: C: C:
4/8 C: 0/1 C: 2/2 (n = 49) 45/49 43/48 12/14 P: 41/41 P: P: P: 0/0
P: 0/1 P: 46/48 46/47 32/35 T-cell malignancies C: 2/2 C: 0/0 C:
0/0 C: 17/17.sup.c C: C: 2/3 (n = 18) P: P: P: P: 1/1 15/16.sup.b
P: 15/16.sup.e 17/18 17/18 P: 1/2 14/15.sup.e Reactive samples C:
0/0 C: 0/0 C: 0/0 C: 0/0 C: 0/0 C: 0/0 (n = 15) P: P: P: P: 14/15
P: P: 15/15 15/15 15/15 15/15 15/15 Miscellaneous (n = 8) C: 3/3 C:
2/2 C: 0/0 C: 3/3 C: 1/1 C: 1/1 P: 3/5 P: 4/6 P: 6/8 P: 5/5.sup.d,d
P: 6/7 P: 5/7 All samples (n = 90) C: C: C: C: 25/29 C: C: 5/6
50/54 45/50 12/14 P: 60/61 16/18 P: P: P: P: P: 80/84 33/36 36/40
70/76 68/72 .sup.aIncludes both VH-JH and DH-JH PCR analysis
.sup.bC, clonal rearrangements; P, polyclonal rearrangements
.sup.cIn one sample clonality in GeneScanning only .sup.dIn one
sample clonality in heteroduplex analysis only .sup.eIn one sample
polyclonality in GeneScanning only .sup.fIn one sample
polyclonality in heteroduplex analysis only
[0427] TABLE-US-00009 TABLE 9 Complementarity of different Ig
multiplex PCR targets for clonality detection in Southern
blot-defined B-cell malignancies Diagnosis.sup.a Pre-germinal
(post-)germinal all B-cell Multiplex PCR center B B-CLL center B
malignancies tubes (n = 8) (n = 16) (n = 25) (n = 49) IGH VH-JH
8/8.sup.b (100%).sup. 14/16.sup.c (88%).sup. 15/25.sup.b (60%)
37/49 (76%) FR1 IGH VH-JH 8/8 (100%) 15/16 (94%) 14/25.sup.b (56%)
37/49 (76%) FR2 IGH VH-JH 8/8 (100%) 14/16 (88%) 11/25.sup.c (44%)
33/49 (67%) FR3 IGH VH-JH 8/8 (100%) 15/16 (94%) .sup. 17/25 (68%)
40/49 (82%) 3FR IGH D.sub.H-J.sub.H 0/8 (0%) 11/16 (69%) .sup.
11/25 (44%) 22/49 (45%) IGH VH-JH + IGH DH-JH 8/8 (100%) 15/16
(94%) .sup. 22/25 (88%) 45/49 (92%) IGK 8/8 (100%) 16/16 (100%)
21/25.sup.d (84%) 45/49 (92%) IGL 4/8 (50%) 7/16.sup.e (44%)
4/25.sup.f (16%) 15/49 (31%) IGH VH-JH + IGK 8/8 (100%) 16/16
(100%) .sup. 21/25 (84%) 45/49 (92%) IGH VH-JH + IGL 8/8 (100%)
15/16 (94%) .sup. 17/25 (68%) 40/49 (82%) IGH VH-JH + IGH 8/8
(100%) 16/16 (100%) .sup. 24/25 (96%) 48/49 (98%) DH-JH + IGK IGH
VH-JH + IGH 8/8 (100%) 16/16 (100%) .sup. 24/25 (96%) 48/49 (98%)
DH-JH + IGK + IGL .sup.aAll samples have clonal gene rearrangements
in at least the IGH locus as determined by Southern blot analysis
.sup.bTwo cases showed clonal products in GeneScanning, but
polyclonal products in heteroduplex analysis .sup.cOne case showed
clonal products in GeneScanning, but polyclonal products in
heteroduplex analysis .sup.dIncluding case 25-NL-4 with weak clonal
IGH but polyclonal IGK gene rearrangements in Southern blot
analysis .sup.eIncluding cases 11-NL-19 and 12-ES-1 with clonal IGH
+ IGK but polyclonal IGL gene rearrangements in Southern blot
analysis
[0428] TABLE-US-00010 TABLE 10 Conditions and control samples for
multiplex PCR analysis of Ig/TCR gene rearrangements and
translocations t(11; 14) and t(14; 18) PCR conditions Multiplex
TaqGold MgCl.sub.2 Positive controls (examples) PCR Tubes Buffer
(U) (mM) polyclonal monoclonal.sup.a IGH VH- A/B/C Gold/ 1 1.5
tonsil A: NALM-6; JH II SU-DHL-5; SU-DHL-6 B: NALM-6; SU-DHL-5;
SU-DHL-6 C: NALM-6; SU-DHL-5; SU-DHL-6 IGH DH- D/E Gold 1 1.5
tonsil D: KCA; ROS15 JH E: HSB-2, HPB- ALL IGK A/B Gold/ 1 1.5
tonsil A: KCA; ROS15 II B: ROS15, 380 IGL A Gold/ 1 2.5 tonsil A:
CLL-1; EB-4B; II KCA TCRB A/B/C II 2 (A, B).sup.b 3.0 (A, B) PB- A:
RPMI-8402; 1 (C) 1.5 (C) MNC.sup.c Jurkat; PEER; DND-41 B: PEER;
CML-T1, MOLT-3 C: Jurkat TCRG A/B II 1 1.5 PB- A: MOLT-3; RPMI-
MNC.sup.c 8402; Jurkat; PEER B: Jurkat; PEER TCRD A II 1 2.0 PB- A:
PEER, REH MNC.sup.c BCL1- A II 1 2.0 NA.sup.c A: JVM 2 IGH BCL2-
A/B/C II 1 1.5 NA.sup.c A: DoHH2; SU- IGH DHL-6 B: K231.sup.d C:
OZ; SC1.sup.d; SU- DHL-16 .sup.aMost clonal cell line controls can
be obtained via the Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH; contact person: dr. H. G. Drexler (address:
Department of Human and Animal Cell Cultures, Mascheroder Weg 1B,
38124 Braunschweig, Germany)..sup.192,193 .sup.bIn most multiplex
tubes only 1 U TaqGold is needed, but 2 U TaqGold are needed in
TCRB tubes A and B because they contain >15 different primers.
.sup.cAbbreviations: PB-MNC, mononuclear cells from peripheral
blood; NA, not applicable. .sup.dThe t(14; 18) positive cell lines
K231, OZ, and SC1 were kindly provided by prof. Martin Dyer,
University of Leicester, Leicester, GB.
[0429] TABLE-US-00011 TABLE 11 Size ranges, non-specific bands, and
detection method in multiplex PCR analysis of Ig/TCR gene
rearrangements and chromosom{dot over (e)} aberrations t(11; 14)
and t(14; 18) GeneScan Preferred running Multiplex Nonspecific
method of time: PCR Size range (bp) bands (bp) analysis
gel/capillary IGH Tube A: 310-360 Tube A: .about.85 GeneScanning
3-3.5 h/ VH-JH Tube B: 250-295 Tube B: -- and heteroduplex 45 min
Tube C: 100-170 Tube C: -- analysis equally suitable IGH Tube D:
110-290 Tube D: 350.sup.a heteroduplex 3-3.5 h/ DH-JH
(D.sub.H1/2/4/5/6-J.sub.H) + 390-420 analysis slightly 45 min
(D.sub.H3-J.sub.H) preferred over GeneScanning Tube E: 100-130 Tube
E: 211.sup.b (variation of product sizes hampers GeneScanning) IGK
Tube A: 120-160 Tube A: -- heteroduplex 3-3.5 h/
(V.kappa.1f/6/V.kappa.7-J.kappa.) + 190-210 analysis slightly 45
min (V.kappa.3f- preferred over J.kappa.) + 260-300 GeneScanning
(V.kappa.2f/V.kappa.4/V.kappa.5- (small junction JK) size +
variation of Tube B: 210-250 Tube B: .about.404 product sizes
V.kappa.1f/6/V.kappa.7-Kde + 270-300 hampers (V.kappa.3f/intron-
GeneScanning) Kde) + 350-390 (V.kappa.2f/V.kappa.4/V.kappa.5- Kde)
IGL Tube A: 140-165 Tube A: -- heteroduplex 2 h/45 min analysis
clearly preferred over GeneScanning (small junction size hampers
GeneScanning) TCRB Tube A: 240-285 Tube A: heteroduplex 2 h/45 min
(273).sup.c analysis slightly Tube B: 240-285 Tube B: preferred
over <150, 221.sup.d GeneScanning Tube C: 170-210 (D.beta.2) +
285-325 Tube C: 128, (limited (D.beta.1) 337.sup.d repertoire,
particularly in case of .psi.V.gamma.10 and .psi.V.gamma.11 usage)
TCRG Tube A: 145-255 Tube A: -- GeneScanning 2 h/45 min Tube B:
80-220 Tube B: -- and heteroduplex analysis equally suitable TCRD
Tube A: 120-280 Tube A: .about.90 heteroduplex 2 h/45 min analysis
clearly preferred over GeneScanning (low amount of template +
variation of product sizes hampers GeneScanning) BCL1- Tube A:
150-2000 Tube A: .about.550 agarose NA.sup.e IGH (weak) BCL2- Tube
A: variable Tube A: -- agarose NA.sup.e IGH Tube B: variable Tube
B: -- Tube C: variable Tube C: -- .sup.aThe nonspecific 350 bp band
is the result of cross-annealing of the D.sub.H2 primer to a
sequence in the region upstream of J.sub.H4. In GeneScanning this
nonspecific band does not comigrate with D-J products (see FIG.
5B). .sup.bThe 211 bp PCR product represents the smallest
background band derived from the germline D.sub.H7-J.sub.H1 region.
When the PCR amplification is very efficient, also longer PCR
products might be obtained because of primer annealing to
downstream J.sub.H gene rearrangements; e.g. 419 bp
(D.sub.H7-J.sub.H2), 1031 bp (D.sub.H7-J.sub.H3), etc. .sup.cThe
273 bp band (mainly visible by GeneScanning) is particularly seen
in samples with low numbers of contaminating lymphoid cells.
.sup.dIntensity of non-specific band depends on primer quality.
.sup.eNA, not applicable
* * * * *
References